H₂S generation rate in <i>lcd</i> plants treated with PEG8000.

<p>Endogenous H₂S generation rate in <i>lcd</i> seedlings treated with 0.2 g ml⁻¹ PEG8000 for 2 h. Results are shown as mean ± SE (n = 3 independent experiments). Letter numbers indicate significant differences between treatmeats and substracts (<i>P<0.05</i>).</p

Effects of PEG8000 on the expression of genes controlling H₂S generation and on H₂S production rate in WT seedlings.

<p>(<b>A</b>) Expression detection of H₂S generating critical enzymes coding genes in WT seedlings treated with 0.1 g ml⁻¹ PEG8000 for 0, 1, 2, 4, 8 h; (<b>B</b>) Expression detection of H₂S generating critical enzymes coding genes in WT seedlings treated 2 h with 0, 0.05, 0.1, 0.2, 0.4 g ml⁻¹ PEG8000. <i>EF1-α</i> was used as an internal control of RT-PCR; (<b>C</b>) Endogenous H₂S production rate of WT seedlings treated with 0.2 g ml⁻¹ PEG8000 for 2 h. Results are shown as mean ± SE (n = 3 independent experiments). Letter numbers indicate significant differences between treatments and substracts (<i>P<0.05</i>).</p

Hydrogen Sulfide Improves Drought Tolerance in <i>Arabidopsis thaliana</i> by MicroRNA Expressions

<div><p>Hydrogen sulfide (H₂S) is a gasotransmitter and plays an important role in many physiological processes in mammals. Studies of its functions in plants are attracting ever growing interest, for example, its ability to enhance drought resistance in <i>Arabidopsis</i>. A general role of microRNAs (miRNAs) in plant adaptive responses to drought stress has thereby increased our interest to delve into the possible interplay between H₂S and miRNAs. Our results showed that treating wild type (WT) <i>Arabidopsis</i> seedlings with polyethylene glycol 8000 (PEG8000) to simulate drought stress caused an increase in production rate of endogenous H₂S; and a significant transcriptional reformation of relevant miRNAs, which were also triggered by exogenous H₂S in WT. When <i>lcd</i> mutants (with lower H₂S production rate than WT) were treated with PEG8000, they showed lower levels of miRNA expression changes than WT. In addition, we detected significant changes in target gene expression of those miRNAs and the corresponding phenotypes in <i>lcd</i>, including less roots, retardation of leaf growth and development and greater superoxide dismutase (SOD) activity under drought stress. We thereby conclude that H₂S can improve drought resistance through regulating drought associated miRNAs in <i>Arabidopsis</i>.</p></div

Summary of H₂S regulating miRNAs in response to drought stress.

<p>Solid lines: direct effects; dotted lines: intermediates remain elusive; arrows: enhanced expression; hyphen: suppressed expression.</p

NaHS effects on miRNAs expression in WT seedlings.

<p>(<b>A</b>) miRNAs expression in WT seedlings treated with 50 µmol L⁻¹ NaHS for 0, 3, 6, 12 h; (<b>B</b>) miRNAs expression in WT seedlings treated with 0, 20, 50, 100 µmol L⁻¹ NaHS for 12 h. <i>EF1-α</i> was used as an internal control.</p

Root number and length in WT and <i>lcd</i> plants treated with PEG8000.

<p>(<b>A</b>) WT and <i>lcd</i> seedlings cultured in ½ MS and ½ MS containing PEG8000 for 26 day. (<b>B</b>) Length of roots. (<b>C</b>) Number of roots. Results are shown as mean ± SE (n = 3 independent experiments). Letter numbers indicate significant differences (<i>P<0.05</i>).</p

PEG8000 and NaHS effects on miRNAs in WT and <i>lcd</i> plants.

<p>(<b>A</b>) <i>MIR167a</i>, <i>MIR167c</i>, <i>MIR167d</i>, <i>MIR393a</i> and <i>MIR396a</i> expressions in WT and <i>lcd</i> treated with 50 µmol L⁻¹ NaHS and 0.2 g ml⁻¹ PEG8000. <i>lcd</i> was pre-treated with 50 µmol L⁻¹ NaHS for 12 h and 0.2 g ml⁻¹ PEG8000 for 2 h; (<b>B</b>) <i>MIR398a</i>, <i>MIR398b</i> and <i>MIR398c</i> expression in WT and <i>lcd</i> treated with 50 µmol L⁻¹ NaHS and 0.2 g ml⁻¹ PEG8000. The same treatments were applied as in (<b>A</b>). <i>ACTIN</i> was used as an internal control in qRT-PCR. Results are shown as mean ± SE (n = 3 independent experiments). Letter numbers indicate significant differences between treatments within one gene (<i>P<0.05</i>).</p

RT-PCR detection of miRNAs transcription in WT seedlings under PEG8000 stress.

<p>(<b>A</b>) miRNAs transcription detection in WT seedlings treated with 0, 1, 2, 4, 8 h using 0.01 g ml⁻¹ PEG8000 treatment; (<b>B</b>) miRNAs transcription detection in WT seedlings after 2 h using different PEG8000 concentration treatments at 0, 0.05, 0.1, 0.2, 0.4 g ml⁻¹ PEG8000. <i>EF1-α</i> was used as an internal control of RT-PCR.</p

Target gene expressions in WT and <i>lcd</i> plants treated with PEG8000 and NaHS.

<p>(<b>A</b>) <i>ARF8</i>, <i>TIR1</i>, <i>AFB2</i>, <i>AFB3</i>, <i>GRF1</i>, <i>GRF2</i> and <i>GRF3</i> expression in WT and <i>lcd</i> treated with 50 µmol L⁻¹ NaHS and 0.2 g ml⁻¹ PEG8000; (<b>B</b>) <i>CSD1</i> and <i>CSD2</i> expression in WT and <i>lcd</i> treated with 50 µmol L⁻¹ NaHS and 0.2 g ml⁻¹ PEG8000. The same treatments were applied as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077047#pone-0077047-g004" target="_blank">Figure 4</a>. <i>ACTIN</i> was used as an internal control in qRT-PCR. Results are shown as mean ± SE (n = 3 independent experiments). Letter numbers indicate significant differences between treatments within one gene (<i>P<0.05</i>).</p

Leaf growth and development of WT and <i>lcd</i> plants treated with PEG8000.

<p>WT and <i>lcd</i> seedlings cultured in ½ MS and ½ MS containing PEG8000 for 26 day. Experiments are repeated three times.</p