Expression of Animal Anti-Apoptotic Gene Ced-9 Enhances Tolerance during <i>Glycine max</i> L.–<i>Bradyrhizobium japonicum</i> Interaction under Saline Stress but Reduces Nodule Formation

<div><p>The mechanisms by which the expression of animal cell death suppressors in economically important plants conferred enhanced stress tolerance are not fully understood. In the present work, the effect of expression of animal antiapoptotic gene Ced-9 in soybean hairy roots was evaluated under root hairs and hairy roots death-inducing stress conditions given by i) <i>Bradyrhizobium japonicum</i> inoculation in presence of 50 mM NaCl, and ii) severe salt stress (150 mM NaCl), for 30 min and 3 h, respectively. We have determined that root hairs death induced by inoculation in presence of 50 mM NaCl showed characteristics of ordered process, with increased ROS generation, MDA and ATP levels, whereas the cell death induced by 150 mM NaCl treatment showed non-ordered or necrotic-like characteristics. The expression of Ced-9 inhibited or at least delayed root hairs death under these treatments. Hairy roots expressing Ced-9 had better homeostasis maintenance, preventing potassium release; increasing the ATP levels and controlling the oxidative damage avoiding the increase of reactive oxygen species production. Even when our results demonstrate a positive effect of animal cell death suppressors in plant cell ionic and redox homeostasis under cell death-inducing conditions, its expression, contrary to expectations, drastically inhibited nodule formation even under control conditions.</p></div

Ced-9 expression inhibits nodule formation in hairy roots.

<p>K599-empty and K599-CED9 composite plants were inoculated with <i>B. japonicum</i> USDA138 and after 14 days the nodulation was evaluated. A) Image of nodules in K599-empty and K599-CED9 hairy roots. B) Number of nodules in K599-empty (black bar) and K599-CED9 (grey bar) composite plants. Total nodules were counted and divided by the total number of hairy roots, including hairy roots without nodules (nodule number per hairy root). C) Nodulated and non-nodulated hairy roots of K599-CED9 composite plants were separated and Ced-9 transgenic hairy roots were identified by Ced-9 PCR (+ indicates Ced-9 positive control). Data are means ± SE of four composite plants. Different letters indicate significant differences between composite plant genotypes (p<0.05, DGC test).</p

Evaluation of malondialdehyde (MDA) and Adenosine-5′-triphosphate (ATP) levels in root hairs.

<p>Two-day soybean seedlings were subjected 30 min to control, inoculated with <i>B. japonicum</i> (inoculated), 50 mM NaCl, inoculated with <i>B. japonicum</i> in presence of 50 mM NaCl (inoc 50 mM NaCl), and 150 mM NaCl conditions. Then, root hairs were extracted from roots and MDA and ATP content were evaluated. Data are means ± SE of four (MDA) and three (ATP) independent experiments (root hairs from 200 roots per experiment). Different letters indicate significant differences between treatments (p<0.05, DGC test).</p

Ced-9 expression ameliorates root hairs death-inducing conditions effects.

<p>K599-empty (A, C, E) and K599-CED9 (B, D, F) hairy roots were subjected 30 min under control (A and B), inoculated with <i>B. japonicum</i> in presence of 50 mM NaCl (inoc 50 mM NaCl) (C and D), and 150 mM NaCl (E and F). Nuclear morphology of root hairs was evaluated. Arrows indicates nuclear fragmentation. Images were taken with a Zeiss confocal microscope. The excitation was performed simultaneously at 488 nm and emission filter BP 500–530 IR and BP 565–615 IR for AO and EtBr, respectively (image overlay).</p

Ced-9 expression prevents ROS generation under hairy roots death-inducing stress conditions.

<p>K599-empty (dark bars) and K599-CED9 (grey bars) hairy roots were subjected 3 h under control, inoculated with <i>B. japonicum</i> in presence of 50 mM NaCl (inoc 50 mM NaCl), and 150 mM NaCl conditions, and redox parameters were evaluated. A) MDA content, B) H₂O₂ content, C) FRAP (Ferric Reducing Ability of Plasma) assay, D) Reduced/total ascorbic acid ratio. Data are means ± SE from five independent hairy roots. Different Latin and Greek letters indicate significant differences between treatments in K599-empty and K599-CED9 hairy roots, respectively (p<0.05, DGC test). Asterisks indicate significant differences between hairy roots genotypes (p<0.05, DGC test).</p

Root hairs death inducing stress conditions.

<p>Two-day soybean seedlings were subjected 30 min to control, inoculated with <i>B. japonicum</i> (inoculated), inoculated with <i>B. japonicum</i> in presence of 50 mM NaCl (inoc 50 mM NaCl), and 150 mM NaCl conditions. A) Evans blue staining of roots showing loss of membrane integrity (upper image) and detail of root hairs staining (image below). B) Evans Blue stain intensity was measured by the image analyzer program Optimas 6.1. C) DNA degradation in a representative sample pool of root and root hairs. 1: Control, 2: inoculated 50 mM NaCl, 3: 150 mM NaCl. 2 µg of DNA were loaded on a 2% TAE agarose gel and stained with ethidium bromide. Data are means ± SE of four independent experiments (two roots per experiment). Different letters indicate significant differences between treatments (p<0.05, DGC test).</p

Additional file 4: of Genome and transcriptome analysis of the Mesoamerican common bean and the role of gene duplications in establishing tissue and temporal specialization of genes

Supplementary datasets S8–S11. (XLS 395 kb

Additional file 5: of Genome and transcriptome analysis of the Mesoamerican common bean and the role of gene duplications in establishing tissue and temporal specialization of genes

Supplementary datasets S12–S24. (XLS 2190 kb

Additional file 3: of Genome and transcriptome analysis of the Mesoamerican common bean and the role of gene duplications in establishing tissue and temporal specialization of genes

Supplementary datasets S5–S7. (XLS 20553 kb