The ectopic expression of <i>GmSAL1</i> in BY-2 cells increased the vacuolar compartmentalization of Na⁺ under NaCl stress.

<p>Four-day-old BY-2 cells grown in MS medium were used, including cells of the untransformed wild type (WT), empty-vector transgenic control (Empty vector), and three independent <i>GmSAL1</i> transgenic lines (A3-1, B3-1, D2-3). Vacuolar Na⁺ compartmentalization was visualized with the use of Sodium Green^TM. Cells pre-washed with MS medium were transferred to fresh MS medium containing no supplements (untreated), MS medium supplemented with 150mM NaCl or 150mM NaCl+ 1μM IP₃ for 1h with shaking. They were then washed with fresh MS medium and stained with 5μM Sodium Green^TM, followed by confocal microscopic analysis. <b>A</b>: Typical photos showing the relative levels of vacuolar Na⁺ using the fluorescent signal of Sodium Green^TM (represented by the pseudo-green color). Scale bar= 50μm.B: Statistical analysis. The relative fluorescence intensity of 17-34 cells (from 4 fields) was determined for each data point. Error bar: standard error. ** indicates a significant difference (p<0.01) between the transgenic lines and WT, based on ANOVA followed by the posthoc Tukey’s test.</p

Ectopic expression of <i>GmSAL1</i> in <i>A. thaliana</i> negated the effects of ABA on stomatal aperture.

<p>Leaves from 4-week-old <i>A. thaliana</i> grown on soil were used in this experiment. <b>A</b>:Representative DIC images of the guard cells of untransformed wild type (WT), empty-vector transgenic control (Empty vector), and two <i>GmSAL1</i> transgenic lines (GmSAL1-1 and GmSAL1-2), treated with 0μM ABA, 100μM ABA, or 100μM ABA + 5mM CaCl_2, were captured using a light microscope. Scale bar =20μm. <b>B</b>: Mean stomatal aperture was measured using a digital ruler. N>168 from repeated experiments. Error bar: standard error. ** indicates a significant difference (p<0.01) between GmSAL1-1 or GmSAL1-2 and WT, based on ANOVA followed by the posthoc Tukey’s test.</p

Aligning GmSAL1 with its closest homologues in <i>A. thaliana</i>.

<p>Multiple alignments were performed for GmSAL1, AtSAL1, and AtSAL2 (Genbank numbers EF637045, Q42546, and NP_201205, respectively), using the ClustalW program [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0078181#B19" target="_blank">19</a>] in the BioEdit package (ver. 7.0.5.3). Identical amino acid residues were shaded black and similar amino acid residues were shaded grey. The conserved motifs involved in substrate- and metal-binding and nucleophilic water activation were marked as A, B and C [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0078181#B38" target="_blank">38</a>].</p

Ectopic expression of <i>GmSAL1</i> in <i>A. thaliana</i> negated the effects of ABA on seed germination.

<p>The germination rates of <i>A. thaliana</i> seeds of untransformed wild type (WT), empty-vector transgenic control (Empty vector), and <i>GmSAL1</i>-expressing lines, treated with 0μM (<b>A</b>), 2.5μM (<b>B</b>), or 4μM (<b>C</b>) ABA on half-strength MS agar plates, were determined. Results were calculated from 138-211 seeds from three independent experiments. Error bar: standard error. The differences of germination rates among WT, empty vector and <i>GmSAL1</i> overexpressing lines were subjected to one-way analysis of variance (ANOVA) followed by the posthoc Tukey’s test. Under 2.5μM ABA treatment, p<0.01 from day 4 to day 7. Under 4μM ABA treatment, p<0.01 from day 4 to day 8.</p

<i>In</i><i>vivo</i> changes in IP₃ signals due to the ectopic expression of <i>GmGAL1</i> in <i>A. thaliana</i>.

<p>The biosensor PH_PLCd was employed to study the changes in IP₃ signals as described in Materials and Methods. Representative fields of the lower epidermis of the original <i>PH</i>_<i>PLCd</i><i>-GFP</i> line and two independent double-transformed <i>A. thaliana</i> lines (<i>PH</i>_<i>PLCd</i><i>-GFP/GmSAL1-1</i> and <i>PH</i>_<i>PLCd</i><i>-GFP/GmSAL1-2</i>) under a confocal microscope were shown. The GFP signal is represented by a pseudo-green color. <b>A</b>: A low magnification showing a wider view, scale bar= 100μm. <b>B</b>: A close-up view of the guard cells, scale bar = 10μm. <b>C</b>:Statistical analysis of the percentage GFP signal in cytoplasm of the guard cells. Results were calculated from 30 cells from 6 fields. Error bar: standard error. ** indicates a significant difference (p<0.01) between the original <i>PH</i>_<i>PLCd</i><i>-GFP</i> construct and the double transformants (<i>PH</i>_<i>PLCd</i><i>-GFP/GmSAL1</i>-1or<i>PH</i>_<i>PLCd</i><i>-GFP/GmSAL1-2</i>), using one-way analysis of variance (ANOVA) followed by the posthoc Tukey’s test. The experiment was performed twice and similar results were obtained.</p

Ectopic expression of <i>GmSAL1</i> in BY-2 cells enhanced their survival rates under NaCl stress.

<p>Four-day-old BY-2 cells grown in MS medium were used, including cells of untransformed wild type (WT), empty-vector transgenic control (Empty vector), and three independent <i>GmSAL1</i> transgenic lines (A3-1, B3-1, D2-3). The survival rate was determined by Trypan blue staining. The cells were untreated, treated with 150mM NaCl or 150mM NaCl+ 1μM IP₃ in MS medium for 24 h with shaking. They were then washed with fresh MS medium and stained with 0.4μg/μl Trypan blue for 15min before microscopic analysis. <b>A</b>: Typical photos showing the rate of survival. The nuclei of dead cells were stained blue. Scale bar= 100μm.B: Statistical analysis. A total of 94-247cells were counted from 4-6 fields. Error bar: standard error. ** indicates a significant difference (p<0.01)between transgenic cells and WT, based on ANOVA followed by the posthoc Tukey’s test.</p

Changes in the protoplast size and vacuolar Na⁺ content under NaCl treatment in real time.

<p><i>GmSAL1</i> transgenic BY-2 cells and the untransformed wild type cells (WT) were treated with 200mM NaCl (see Materials and Methods). Differential interference contrast (DIC) images and Sodium Green^TM fluorescent signals were collected by confocal microscopy (see Materials and Methods), over a period of 50min. Each data point represents the average value of 4-5 cells. Closed square: Na⁺ content of the <i>GmSAL1</i> transgenic line; closed triangle: Na⁺ content of WT; open square: protoplast size of the <i>GmSAL1</i> transgenic line; open triangle: protoplast size of the untransformed wild type (WT). Images were collated to produce the Video S1. The differences in signal intensity and cell size between WT and <i>GmSAL1</i> transgenic lines were subjected to the Student’s T-test. For Sodium Green signal comparison, p<0.01 from 20 sec after NaCl treatment till the end of the experiment (50 min and 20 sec). For cell size comparison, p<0.05 from 25 min and 20 sec after NaCl treatment till the end of the experiment.</p

GmSAL1 Hydrolyzes Inositol-1,4,5-Trisphosphate and Regulates Stomatal Closure in Detached Leaves and Ion Compartmentalization in Plant Cells

<div><p>Inositol polyphosphatases are important regulators since they control the catabolism of phosphoinositol derivatives, which are often signaling molecules for cellular processes. Here we report on the characterization of one of their members in soybean, GmSAL1. In contrast to the substrate specificity of its <i>Arabidopsis</i> homologues (AtSAL1 and AtSAL2), GmSAL1 only hydrolyzes inositol-1,4,5-trisphosphate (IP₃) but not inositol-1,3,4-trisphosphate or inositol-1,4-bisphosphate.The ectopic expression of <i>GmSAL1</i> in transgenic <i>Arabidopsis thaliana</i> led to a reduction in IP₃ signals, which was inferred from the reduction in the cytoplasmic signals of the <i>in vivo</i> biomarker pleckstrin homology domain–green florescent protein fusion protein and the suppression of abscisic acid-induced stomatal closure. At the cellular level, the ectopic expression of <i>GmSAL1</i> in transgenic BY-2 cells enhanced vacuolar Na⁺ compartmentalization and therefore could partially alleviate salinity stress.</p> </div