<i>AtMIR393A</i> and <i>AtMIR393B</i> are partially redundant for proper leaf morphogenesis.

<p>(A) Schematic representation of the normal range of cotyledon epinasty (top) and the extreme cotyledon epinasty (bottom) typical of the auxin-hypersensitive response. (B) The incidence of cotyledon auxin-hypersensitive response in populations of Col-0 (open bars), <i>mir393a-1</i> (light grey bars), <i>mir393b-1</i> (dark grey bars), and <i>mir393ab</i> double mutants (dark bars). Seedlings (n>40 for each condition and genotype) were grown on media containing the concentration of NPA indicated and harvested 4 d after germination. <i>P</i> values (two-tailed Fisher's exact test) for significant differences towards Col-0 are indicated; NS for <i>P</i>>0.05, * for <i>P</i>≤0.05, ** for <i>P</i>≤0.01, *** for <i>P</i>≤0.001, **** for <i>P</i>≤0.0001. <i>P</i> values for significant differences between mutants are given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095972#pone.0095972.s002" target="_blank">Fig. S2</a>. (C) Epinasty of leaf number 1 to 8 for Col-0, <i>mir393a-1</i>, <i>mir393b-1</i> and <i>mir393ab</i> was measured by the vertical distance between the adaxial leaf side and the leaf margin (in mm ± SEM) (see drawing in the insert). Significant difference towards Col-0 is indicated (two-tailed student <i>t</i>-test). * for <i>P</i>≤0.05, ** for <i>P</i>≤0.01. N = 10. <i>P</i> values for significant differences between mutants are given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095972#pone.0095972.s002" target="_blank">Fig. S2</a>.</p

miR393 is required for proper degradation of AXR3-NT:GUS proteins.

<p>Representative pictures of wt and <i>mir393</i> mutant roots expressing the <i>HSpro:AXR3-NT:GUS</i> gene. Without treatments, the AXR3-NT:GUS fusion protein is more stable in <i>mir393a-1</i>, <i>mir393b-1</i> and <i>mir393ab</i> mutants compared to wt plants. Short treatments with 10 µM auxin induces the degradation of AXR3-NT:GUS, while treatment with 20 µM auxinole or MG132 blocks the degradation of AXR3-NT:GUS. This shows that the SCF complexes and proteasome are functional in the mutants as well. Clearance of presumably high levels of endogenous AUX/IAA proteins by treatment with 2,4-D for 4 h restores the proper degradation of AXR3-NT:GUS.</p

Identification and characterization of <i>MIR393A</i> T-DNA insertion mutants.

<p>(A) Schematic map showing the 5′-3′ orientation of genes (large arrows and gene names are indicated) flanking <i>AtMIR393A</i> on chromosome II. The arrow representing <i>AtMIR393A</i> indicates the pri-miRNAs which full-length sequence determined by RACE experiments (given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095972#pone.0095972.s001" target="_blank">Fig. S1</a>). The expanded region represents the folded pre-miR393 nucleotide sequence with the miR393 sequence indicated in red. The open triangle represents the T-DNA insertion. The scheme is not drawn to scale. (B) RNA-blot hybridization of RNA prepared from roots and leaves of 34d-old plants. Probed RNAs are indicated on the right. The signal detected for mutants relative to wild-type Col-0 are normalized relative to the <i>Midori Green</i> stained RNA signals. Similar results were obtained in three independent experiments.</p

Basal expression level of primary auxin-inducible <i>AUX/IAA</i> genes in <i>mir393</i> mutants compared to wt plants.

<p>Pictures of representative wt and <i>mir393</i> mutant plants expressing the <i>DR5pro:GUS</i> (A), the <i>IAA12pro:GUS</i> (B), or the <i>IAA3pro:GUS</i> gene (C) upon treatment or not with 10 µM 2,4-D for 8 h. Arrows highlight the GUS staining detected.</p

miR393 is required for proper induction of <i>AUX/IAA</i> and <i>GH3</i> genes.

<p>Northern blots of RNAs from wt and <i>mir393ab</i> mutant plantlets treated or not with 10 µM 2,4-D for 8 h. Without treatments, <i>IAA17</i> and <i>IAA19</i> have higher steady state levels in <i>mir393ab</i> mutants than in wt plants. Treatment with 2,4-D for 8 h induces the expression of all genes tested. <i>GH3.5</i> accumulates to similar levels in wt and mutants, <i>GH3.2</i>, <i>IAA19</i>, <i>IAA29</i>, <i>IAA5</i> and <i>IAA2</i> accumulate to higher levels in <i>mir393ab</i> mutants than in wt plants while <i>GH3.6</i> and <i>IAA17</i> accumulate to lower levels in <i>mir393ab</i> mutants than in wt plants.</p

NaCl influences auxin-dependent morphological responses through miR393 regulation.

<p>Four-dpg WT and <i>mir393ab</i> seedlings were transferred onto ATS medium containing 75 mM NaCl. Arrowheads mark the position of the root tip at the time of transfer from standard to treatment conditions. Representative photographs of seedlings after 5 d of treatment are shown in (<b>A</b>) LR were quantified at designed times (<b>B</b>) Data are mean values (±SE) of three independent experiments. (<b>C</b>) and (<b>D</b>) Quantification of emergent and mature LR in root region A and region B after transfer to standard or salt stress conditions, respectively. Data are mean values (±SE) of three independent experiments. Different letters indicate a significant difference at P≤0.05 (Tukey test). (<b>E</b>) Suppression of LR and PR growth under 75 mM NaCl conditions measured as a percentage of growth relative to standard conditions. Seedlings were transferred to salt at 4 dpg and grown for 5 d post treatment. Data are mean values (±SE) of three independent experiments. Asterisks mark significant changes between WT and <i>mir393ab</i> at P≤0.05 (<i>t</i>- test). PR: WT −NaCl 7.35 cm+/−0.55; WT +NaCl 4.34 cm+/−0.39; <i>mir393ab</i> −NaCl 7.41 cm+/−0.29; <i>mir393ab</i> +NaCl 5.5 cm+/−0.17. LR: WT −NaCl 0.95 cm+/−0.15; WT +NaCl 0.39 cm+/−0.12; <i>mir393ab</i> −NaCl 0.96 cm+/−0.14; <i>mir393ab</i> +NaCl 0.57 cm+/−0.08…</p

Auxin responses are affected by salinity in Arabidopsis seedlings.

<p>(<b>A</b>) Four dpg WT seedlings were transferred from auxin-free medium onto ATS medium containing no auxin or 85 nM IAA in combination with increasing concentrations of NaCl. The total number of emerged lateral roots was counted 4 d after the transfer to new media. Data are mean values (±SE) of three independent experiments. Different letters indicate a significant difference at P≤0.05 (Tukey test). (<b>B</b>) Seven dpg <i>BA3pro:GUS</i> seedlings were incubated on 50 nM or 100 nM IAA in combination with increasing concentrations of NaCl for 4 h. GUS activity was revealed after incubation with X-Gluc at 37°C. GUS staining in representative root tip segment is shown. (<b>C</b>) Relative transcript level of GUS upon 100 nM IAA treatment in combination with NaCl as described in (B). The control value is arbitrarily set to 1 in each case. Data are mean values (±SE) of three independent experiments. Different letters indicate a significant difference at P≤0.05 (Tukey test). (<b>D</b>) Seven dpg <i>DR5pro:GUS</i> seedlings were incubated with 200 mM NaCl for 4 h and subjected to GUS staining. Images show different parts of seedlings: cotyledons (Cotyl), shoots, root tip and LR. The GUS signal detected in NaCl treatment relative to control is shown. The control value is arbitrarily set to 1 in each case. Data are mean values of three independent experiments.</p

Salt-mediated down-regulation of TIR1 by miRNAs.

<p>(<b>A</b>) Seven dpg WT, <i>ago1-27</i> and <i>mir393ab</i> seedlings were subjected to 200 mM NaCl treatment for 4 h. Relative transcript level of <i>TIR1</i> upon treatment was measured by RT-PCR. The control value is arbitrarily set to 1 in each case. Data are mean values (±SE) of three independent experiments. Different letters indicate a significant difference at P≤0.05 (Tukey test). (<b>B</b>) Seven dpg <i>TIR1pro:TIR1-GUS</i> and <i>TIR1pro:mTIR1-GUS</i> seedlings were incubated in liquid ATS medium with NaCl for 4 h and then subjected to GUS staining. Representative photographs of root tips are shown. The control value is arbitrarily set to 1 in each case. Data are mean values of three independent experiments. (<b>C,D</b>) Seven dpg <i>AtMIR393Apro:GUS</i> seedlings were transferred to liquid ATS medium supplemented with increasing concentrations of NaCl for 2 h. GUS activity was revealed after incubation with X-Gluc at 37°C. GUS staining in representative leaves and root segments are shown. The control value is arbitrarily set to 1 in each case. Data are mean values of three independent experiments. (<b>E</b>) Relative transcript level of GUS in NaCl- treated <i>AtMIR393Apro:GUS</i> seedlings was quantified upon treatment. The control value is arbitrarily set to 1 in each case. Data are mean values (±SE) of three independent experiments. Different letters indicate a significant difference at P≤0.05 (Tukey test).</p

miR393 early regulates redox components under salt.

<p>Seven dpg WT and <i>mir393ab</i> seedlings were transferred onto liquid ATS medium supplemented with 100 mM NaCl. After 12 h of initial treatment (<b>A</b>) H₂O₂ accumulation, (<b>B</b>) APX activity and (<b>C</b>) CAT activity were measured. (<b>D</b>) Seven dpg <i>mir393ab</i>, <i>tir1-1</i>, <i>tir1-1 35Spro:TIR-Myc</i> and WT seedlings were treated with 100 mM NaCl for 3 d. Chlorophyll content was measured and expressed as percentage of untreated seedlings. Data are mean values (±SE) of three independent experiments. Different letters indicate a significant difference at P≤0.05 (Tukey test).</p

Modulation of plant development and acclimation to salinity are interconnected responses influenced by miR393 regulation node.

<p>These two processes are strongly regulated by reciprocal interaction between ROS and auxin signaling pathway. NaCl-mediated stress would conduct to miR393 induction that targets TIR1-AFB2 auxin receptors leading to stabilization of Aux/IAA repressors and down- regulation of auxin signaling pathway. In turn, auxin modulation influences on ROS-associated metabolism. Salt exposition leading to SIMR would be under negative regulation of the auxin signaling pathway. Depending on time and intensity of salt stress, early ROS-auxin crosstalk may exert impact on SIMR and tolerance to salinity. Others hormones and/or miRNAs may also regulate auxin-dependent pathway and physiological responses during acclimation to salinity.</p