Effects of GSH, BSO and HRW alone or their combinations on ascorbic acid pool (AsA and DHA) in the roots of <i>Brassica campestris</i> under Cd or none stress.

<p>The germinated seeds were grown in nutrient solution for one day and then shifted to solutions with 0 or 50 μM CdCl₂ for another 12 h. After the removal of Cd stresses, the seedlings were then incubated in solution with 25 μM GSH, 10 μM BSO or 50% saturation of HRW alone or their combinations for 48 h. Data are means ± SE from three independent experiments. Bars with different letters are significantly different at <i>P</i><0.05 according to Duncan’s multiple range test.</p

HRW post-treatment alleviated Cd stress-induced <i>Brassica campestris</i> seedlings growth inhibition.

<p>The germinated seeds were grown in nutrient solution for one day and then shifted to solutions with 0 or 50 μM CdCl₂ for another 12 h. After the removal of Cd stresses, the seedlings were then incubated in solution with H₂O or 50% saturation of HRW for 5 days. Bar = 3 cm (A). Changes in root length (B) and fresh weight (C) during the 5 days post-treated with H₂O or 50% saturation of HRW. Data are means ± SE from three independent experiments. Bars with different letters are significantly different at <i>P</i><0.05 according to Duncan’s multiple range test.</p

Representative 2-DE maps of the rice B1139 and B1195 roots copper-binding proteins eluted from a Cu-IMAC column with elution buffer containing 40 mmol imidazole.

<p>The 7 days seedlings were treated with 8 μmol Cu²⁺ for 3 days. Seedlings without any treatment, grown in a full-strength nutrient solution were used as a control (0.32 μmol Cu²⁺). 100 μg of copper-binding protein were loaded onto IPG dry strips (17 cm, pH 4–7 linear gradient; Bio-Rad) during the rehydration step (13 h), followed by focusing for a total of 6,000 V·h using a PROTEAN IEF CELL (Bio-Rad). SDS-PAGE in the second dimension was carried out using 12% SDS—polyacrylamide gels. The protein spots were visualized by mass spectrometry-compatible silver staining.</p

Potential Cu-binding motifs of identified proteins.

<p>Note:—indicates not present; X represents any amino acid, C represents cysteine, H represents histidine, M represents methionine;</p><p>^a Motifs that were reported by Smith et al.;</p><p>^b Motifs that were reported by Kung et al</p><p>Potential Cu-binding motifs of identified proteins.</p

Cadmium-Induced Hydrogen Accumulation Is Involved in Cadmium Tolerance in <i>Brassica campestris</i> by Reestablishment of Reduced Glutathione Homeostasis

<div><p>Hydrogen gas (H₂) was recently proposed as a therapeutic antioxidant and signaling molecule in clinical trials. However, the underlying physiological roles of H₂ in plants remain unclear. In the present study, hydrogen-rich water (HRW) was used to characterize the physiological roles of H₂ in enhancing the tolerance of <i>Brassica campestris</i> against cadmium (Cd). The results showed that both 50 μM CdCl₂ and 50%-saturated HRW induced an increase of endogenous H₂ in <i>Brassica campestris</i> seedlings, and HRW alleviated Cd toxicity related to growth inhibition and oxidative damage. Seedlings supplied with HRW exhibited increased root length and reduced lipid peroxidation, similar to plants receiving GSH post-treatment. Additionally, seedlings post-treated with HRW accumulated higher levels of reduced glutathione (GSH) and ascorbic acid (AsA) and showed increased GST and GPX activities in roots. Molecular evidence illustrated that the expression of genes such as <i>GS</i>, <i>GR1</i> and <i>GR2</i>, which were down-regulated following the addition of Cd, GSH or BSO, could be reversed to varying degrees by the addition of HRW. Based on these results, it could be proposed that H₂ might be an important regulator for enhancing the tolerance of <i>Brassica campestris</i> seedlings against Cd, mainly by governing reduced glutathione homeostasis.</p></div

Effects of GSH, BSO and HRW alone or their combinations on Cd-induced inhibition of root elongation in <i>Brassica campestris</i> seedlings.

<p>The germinated seeds were grown in nutrient solution for one day and then shifted to solutions with 0 or 50 μM CdCl₂ for another 12 h. After the removal of Cd stresses, the seedlings were then incubated in solution with 25 μM GSH, 10 μM BSO or 50% saturation of HRW alone or their combinations for 48 h. Data are means ± SE from three independent experiments. Bars with different letters are significantly different at <i>P</i><0.05 according to Duncan’s multiple range test.</p

Effects of GSH, BSO and HRW alone or their combinations on gene expression of GCS (A), GS(B), GR(C), GST (D) and GPX (E) activities in the roots of <i>Brassica campestris</i> upon Cd or none stress.

<p>The germinated seeds were grown in nutrient solution for one day and then shifted to solutions with 0 or 50 μM CdCl₂ for another 12 h. After the removal of Cd stresses, the seedlings were then incubated in solution with 25 μM GSH, 10 μM BSO or 50% saturation of HRW alone or their combinations for 48 h. Data are means ± SE from three independent experiments. Bars with different letters are significantly different at <i>P</i><0.05 according to Duncan’s multiple range test.</p

Schematic representation of the pathway induced by cadmium including the participation of H₂, GSH and AsA.

<p>Schematic representation of the pathway induced by cadmium including the participation of H₂, GSH and AsA.</p

Effects of GSH, BSO and HRW alone or their combinations on loss of plasma membrane integrity (A), reactive oxygen species (ROS) localization (B) and TBARS (C) in the roots of <i>Brassica campestris</i> under Cd or none stress.

<p>The germinated seeds were grown in nutrient solution for one day and then shifted to solutions with 0 or 50 μM CdCl₂ for another 12 h. After the removal of Cd stresses, the seedlings were then incubated in solution with 25 μM GSH, 10 μM BSO or 50% saturation of HRW alone or their combinations for 48 h. Data are means ± SE from three independent experiments. Bars with different letters are significantly different at <i>P</i><0.05 according to Duncan’s multiple range test.</p

An increase in hydrogen (H₂) evolution in <i>Brassica campestris</i> seedlings upon CdCl₂ and HRW treatment.

<p>The germinated seeds were grown in nutrient solution for one day and then shifted to solutions with 0 or 50 μM CdCl₂ for another 12 h. After the removal of Cd stresses, the seedlings were then incubated in solution with H₂O or 50% saturation of HRW for 48 h. Data are means ± SE from three independent experiments. Bars with different letters are significantly different at <i>P</i><0.05 according to Duncan’s multiple range test.</p