Performance comparison.

<p>Performance comparison.</p

Osmotic tolerance and detached leaf water-loss rates of <i>AtRDUF1</i> overexpression plants.

<p>(A) WT, <i>atrduf1atrduf2</i>, <i>atrduf1</i>, RDUF1-L1, RDUF1-L2 and RDUF1-L3 with or without osmotic stress treatment. 3-day-old seedlings were transferred to 1/2 MS medium containing 0, 200, or 350 mM mannitol, and vertically cultured for 6 d. Bars represent 1 cm. (B) Statistical comparison of root lengths of seedlings under the conditions described in (A). (C) Water loss rates of detached leaves. Detached rosette leaves from wild-type and <i>AtRDUF1</i> overexpression seedlings were incubated for 6 h at room temperature. Water-loss rate is calculated as the ratio between water loss and plant initial fresh weight (FW), expressed in %. Data are presented as means ± SD. Asterisks indicate significance (*, P<0.05 versus WT control).</p

The E3 Ligase AtRDUF1 Positively Regulates Salt Stress Responses in <i>Arabidopsis thaliana</i>

<div><p>Ubiquitination is an important post-translational protein modification that is known to play critical roles in diverse biological processes in eukaryotes. The RING E3 ligases function in ubiquitination pathways, and are involved in a large diversity of physiological processes in higher plants. The RING domain-containing E3 ligase AtRDUF1 was previously identified as a positive regulator of ABA-mediated dehydration stress response in <i>Arabidopsis</i>. In this study, we report that AtRDUF1 is involved in plant responses to salt stress. <i>AtRDUF1</i> expression is upregulated by salt treatment. Overexpression of <i>AtRDUF1</i> in <i>Arabidopsis</i> results in an insensitivity to salt and osmotic stresses during germination and seedling growth. A double knock-out mutant of <i>AtRDUF1</i> and its close homolog <i>AtRDUF2</i> (<i>atrduf1atrduf2</i>) was hypersensitive to salt treatment. The expression levels of the stress-response genes <i>RD29B</i>, <i>RD22</i>, and <i>KIN1</i> are more sensitive to salt treatment in <i>AtRDUF1</i> overexpression plants. In summary, our data show that <i>AtRDUF1</i> positively regulates responses to salt stress in <i>Arabidopsis</i>.</p></div

Histochemical localization of GUS activity in <i>AtRDUF1::GUS</i> transgenic plants.

<p>(A) Developing seed at 12 days after pollination. (B) Desiccated mature seed. (C) Broken mature seed. (D) Dissected seed after imbibition. (E–H) Germinating seedlings at 1-day (E), 2-days (F), 3-days (G), and 4-days (H) after germination. (I) Leaf. (J) Root. (K) Flower. (L) Pollen. (M) Siliques. Bars represent 0.25 mm in A-D; 0.1 mm in J; 10 μm in L; and 1 mm in E-I, K, and M.</p

Cytokine and chemokine expression profiles from monocytes modulated by rD-7.

<p>Supernatants of monocytes stimulated with rD-7 or rD-7/D from three donors were used to assess their cytokine and chemokine expression using Proteome Profiler R&D Systems according to the manufacturer’s instructions. The experiments were performed in duplicate using monocytes from three of the four donor samples shown in Fig. 6. Results of one of three donor samples are shown in A and the rest are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090999#pone.0090999.s003" target="_blank">Fig. S3</a> (*, P<0.05; **, P<0.01, ***, P<0.001). The mean pixel density of dots shown in B were obtained using Odyssey Imaging System using data from all three donors.</p

CD206, CD80 and CD86 expression on CD14+ cells after stimulation with rD-7 and r6–8.

<p>(A) 1×10⁶ monocytes were treated with rD-7 (2.5 µg/ml), r6–8 (2.5 µg/ml) or LPS (2 µg/ml) for 24 h, cells collected and live cells sorted using the vital dye eFluor 780. CD14+ cells were then gated for the analysis of CD206, CD80 and CD86 expression by flow cytometry. Black and grey histograms represent stained cells and unstained cells respectively. (B) The MFI of CD14+ cells expressing CD206, CD80 or CD86 are shown. rD-7, r6–8 or LPS was used in three independent experiments at least and each dot represents a separate experiment (*, P<0.05; ***, P<0.001).</p

CD206 expression on CD14+ cells after stimulation with digested rD-7.

<p>(A) Monocytes were treated with rD-7 (2.5 µg/ml), or PK-digested rD-7 for 24 h and CD206 expression on CD14+ cells was analysed as described in legend to Fig. 1. Black and grey histograms represent stained cells and unstained cells respectively. (B) The MFI of CD14+ cells expressing CD206 are shown.rD-7 or digested rD-7 was used in three independent experiments at least and each dot represents a separate experiment (***, P<0.001).</p

Analyses of CEACAM1 expression on CD14+ monocytes.

<p>(A) Monocytes were incubated with rabbit anti-CEACAM antibody A0115 (black histogram) or its IgG control (grey histogram) followed by secondary PE-conjugated anti-rabbit IgG and FITC-conjugated CD14 mAb. Dotted histogram represents secondary antibody only control. (B) Cells were also labelled with PE-conjugated anti-CEACAM1 mAb (R and D Systems MAb2244) (black histogram) or its isotype control (grey histogram) and FITC-conjugated CD14 mAb. Live CD14+ cells were analysed and the percentages of cells expressing CEACAM1 were found to be 7.2±5.1% using A0115 and 13.9±4.1% using MAb2244. The MFI plots of CEACAMs on CD14+ monocytes are shown on the right (*, P<0.05; **, P<0.01).The three dots in each case represent data from monocytes obtained from three donors. Note the differences in the values shown for the two antibodies could also be assigned to the experiments being performed using two different instruments (Canto II, University of Bristol and Calibur, Shandong University respectively) using different settings.</p

TNF-α, IL-6, IL-10 and IL-12p70 from monocytes modulated by rD-7 or rD-7/D.

<p>Supernatants of 1×10⁶ monocytes stimulated with rD-7 (2.5 µg/ml), rD-7/D (2.5 µg/ml) or LPS (2 µg/ml) for 24 h were collected and TNF-α (A), IL-6 (B), IL-10 (C) and IL-12p70 (D) secretion was determined by using Luminex assay kit. Each dot represents the mean of triplicate estimations within an experiment and the four dots in each case represent data from monocytes obtained from four donors (*, P<0.05; **, P<0.01, ***, P<0.001).</p

CD206 expression on CD14+ cells after stimulation with rD-7/D.

<p>(A) Monocytes treated with rD-7, rD-7/D, or PK-digested samples of the proteins (2.5 µg/ml each) were collected after 24 h, live CD14+ cells were gated and CD206 expression was analysed. Black and grey histograms represent stained and unstained cells respectively. (B) The MFI of CD14+ cells expressing CD206 are shown. rD-7, rD-7/D, LPS, digested rD-7, digested rD-7/D or Proteinase k was used in three independent experiments at least and each dot represents a separate experiment. (C) Surface binding of rD-7 to CD15+ cells. 1×10⁶ CD15+ cells were blocked with A0115 or its IgG control (50 µg/ml) for 30 min on ice, the cells were then stimulated with rD-7 or rD-7/D (2.5 µg/ml each) for 1 h on ice, rD-7 bound to live CD15+ cells was detected with primary mouse anti rD-7 antiserum and secondary PE-conjugated anti mouse IgG Abs by flow cytometry. (*, P<0. 05; **, P<0. 01; ***, P<0. 001).</p