Untitled Item

Hemataxylin and Evans blue staining of rice roots of two contrasting varieties under Al stres

Effect of salt stress on germination efficiency and root growth of transgenic <i>Arabidopsis</i> lines.

<p>(A) The wildtype (WT, col-0) and transgenic (line 1, 35S::<i>VrNHX1</i> and line 4, RD29A::<i>VrNHX1</i>) seedlings were observed for germination score after 10 days exposure to salt stress (150 mM NaCl). (B) Root growth inhibition in wild type (WT, Col-0) and transgenic <i>Arabidopsis</i> (Line 1, 35S::<i>VrNHX1</i> and Line 4, RD29A::<i>VrNHX1</i>) plants upon salt stress (150 mM NaCl) was studied. The 4 days old germinated seedlings were transferred to 150 mM NaCl stress for a period of 7 days and (C) root length measured was plotted in graph. Values indicate means ± SE (n = 10). Statistically significant values at P≤0.05 are indicated as “*”, using Bonferroni analysis.</p

Total intracellular ion measurement in leaves and roots of early and mid stage mungbean seedlings.

<p>Na⁺ and K⁺ content in (A) leaves and (B) roots of unstressed and salt stressed mungbean seedlings harvested at time intervals of 0, 6, 12, 24, 48, and 72 hrs was measured using Flame Photometer. Values indicate means ± SE (n = 3). Statistically significant values at P≤0.05 are indicated as “*”, using Bonferroni analysis.</p

The phylogenetic tree for plant Na⁺/H⁺ antiporters was generated using MEGA4: Tree Explorer software.

<p>The evolutionary history was inferred using the neighbor-joining method and analyzed using bootstrap analysis with 500 replicates. Branches corresponding to partitions reproduced in less than 50% bootstrap replicates are collapsed. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Poisson correction method and are in the units of the number of amino acid substitutions per site. The GenBank Accession numbers for NHX proteins used are: VrNHX1 (AEO50758.1), VuNHX1 (AEO72079.2), GmNHX1 (AAY430061.1), CkNHX1 (ABG89337.1), MsNHX1 (AAS84487.1), CaNHX1 (ADL28385.1), TrNHX1 (ABV00895.1), LtNHX1 (ACE78322.1), AhNHX1 (ADK74832.1), AtNHX1 (NM_122597.2).</p

Studying the physiological changes in transgenic <i>Arabidopsis</i> lines under salt stress.

<p>(A) Effect of salt stress in wild type (WT, Col-0) and transgenic <i>Arabidopsis</i> lines expressing <i>VrNHX1</i> constitutively (Lines 1–3, 35S::<i>VrNHX1</i>) and inducibly (Lines 4–6, RD29A::<i>VrNHX1</i>). NaCl-induced morphological changes was visible in 10 days old WT and transgenic lines after exposure to 200 mM NaCl for 5 days. (B) Changes in chlorophyll, MDA and proline content were estimated and analyzed as explained in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106678#s2" target="_blank">materials and methods</a> section. Values indicate means ± SE (n = 3). Statistically significant values at P≤0.05 are indicated as “*”, using Bonferroni analysis.</p

Copy number analysis of <i>VrNHX1</i> in mungbean genome.

<p>Mungbean genomic DNA (20 µg) was digested with EcoRI and HindIII, and hybridized with DIG-labeled probe corresponding to the CDS of <i>VrNHX1</i>. Hybridization signals are indicated as arrows.</p

Cation sensitivity assay of transformed yeast strains (W303-1B, AXTYES2.0, AXTVrNHX1) under various concentrations of NaCl, KCl, and LiCl.

<p>Saturated seed cultures for each strain was diluted to an OD₆₀₀ of 0.006 and inoculated to liquid APGal medium (pH 5.5) supplemented with or without various concentrations of (A) NaCl (0, 50, 75, 100 mM), B) KCl (0, 0.5, 0.75, 1.0 M), and (C) LiCl (0, 15, 20, 25 mM). Growth was observed at 30°C after 3 days and absorbance recorded at 600 nm. Data are means of 3 independent events (n = 3) and standard errors are plotted in the graph. Statistically significant values at P≤0.05 are indicated as “*”, using Bonferroni analysis.</p

Salt tolerance assay in mature transgenic <i>Arabidopsis</i> lines under salt stress.

<p>(A) Effect of salt stress on wild type (WT, Col-0) and transgenic <i>Arabidopsis</i> lines expressing <i>VrNHX1</i> constitutively (Line 1, 35S::<i>VrNHX1</i>) and inducibly (Line 4, RD29A::<i>VrNHX1</i>) subjected to 250 mM NaCl treatment for 2 weeks (B) Relative transgene expression level of <i>VrNHX1</i> in transgenic <i>Arabidopsis</i> lines under unstressed and salt stressed conditions. No transgene expression was observed in WT. A 0.283 kb fragment of <i>VrNHX1</i>::35SployA and 0.150 kb fragment of <i>AtUBQ1</i> was amplified in quantitative RT-PCR analysis (C) Na⁺ and (D) K⁺ content (µmoles/g DW) was estimated in leaves of unstressed (0 mM NaCl) and salt stressed (250 mM NaCl) WT and transgenic lines, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106678#s2" target="_blank">materials and methods</a>. Values indicate means ± SE (n = 3). Statistically significant values at P≤0.05 are indicated as “*”, using Bonferroni analysis.</p

Total intracellular ion estimation in yeast strains W303-1B, AXTYES2.0 and AXTVrNHX1.

<p>Yeast cells were grown in APG medium (pH 4.0) with 1 mM KCl supplemented in presence (stressed) or absence of 75 mM NaCl (unstressed) and harvested at a cell density of 0.3. Total intracellular, vacuolar and cytoplasmic Na⁺ and K⁺ content was determined as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106678#s2" target="_blank">materials and methods</a> section. Data are means of 3 independent events (n = 3) and standard errors are plotted in the graph. Statistically significant values at P≤0.05 are indicated as “*”, using Bonferroni analysis.</p

Expression analysis of <i>Os</i>NRAT1 under stressed and unstressed condition.

<p>Expression analysis of <i>Os</i>NRAT1 under stressed and unstressed condition.</p