Figure 1.
Auxin responses are affected by salinity in Arabidopsis seedlings.
(A) 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) Seven dpg BA3pro:GUS 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. (C) 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). (D) Seven dpg DR5pro:GUS 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.
Figure 2.
Salinity represses auxin signaling pathway.
Seven dpg TIR1pro:TIR1-GUS (A) and AFB2pro:AFB2-GUS (B) seedlings were incubated in liquid ATS medium with increasing concentrations of 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. (C) Seven dpg tir1-1 35S::TIR1-Myc plants were transferred to liquid ATS medium supplemented with 200 mM NaCl for different times. Total proteins were extracted and protein blot analysis was carried out using an anti cMyc antibody (upper panel). Ponceau staining of Rubisco is shown (bottom panel). (D) Densitometric analysis of three independent immunoblots as indicated in (C). Data are mean values (±SE). Different letters indicate a significant difference at P≤0.05 (Tukey test). (E) Seven dpg HSpro:AXR3NT-GUS seedlings were transferred for 2 h to 37°C and then incubated with 100 and 200 mM NaCl in the absence (upper panel) or presence (lower panel) of IAA. GUS activity was revealed after incubation with X-Gluc at 37°C. Representative photographs and 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.
Figure 3.
Salt-mediated down-regulation of TIR1 by miRNAs.
(A) Seven dpg WT, ago1-27 and mir393ab seedlings were subjected to 200 mM NaCl treatment for 4 h. Relative transcript level of TIR1 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) Seven dpg TIR1pro:TIR1-GUS and TIR1pro:mTIR1-GUS 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. (C,D) Seven dpg AtMIR393Apro:GUS 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. (E) Relative transcript level of GUS in NaCl- treated AtMIR393Apro:GUS 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).
Figure 4.
NaCl influences auxin-dependent morphological responses through miR393 regulation.
Four-dpg WT and mir393ab 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 (A) LR were quantified at designed times (B) Data are mean values (±SE) of three independent experiments. (C) and (D) 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). (E) 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 mir393ab at P≤0.05 (t- test). PR: WT −NaCl 7.35 cm+/−0.55; WT +NaCl 4.34 cm+/−0.39; mir393ab −NaCl 7.41 cm+/−0.29; mir393ab +NaCl 5.5 cm+/−0.17. LR: WT −NaCl 0.95 cm+/−0.15; WT +NaCl 0.39 cm+/−0.12; mir393ab −NaCl 0.96 cm+/−0.14; mir393ab +NaCl 0.57 cm+/−0.08…
Figure 5.
ROS accumulation in LR of mir393ab seedlings during acclimation to salinity.
Four dpg WT and mir393ab seedlings were transferred onto ATS medium containing 75 mM NaCl. After 5 d of treatment, endogenous H2O2 was detected in situ by H2DCF DA probe. Quantification of probe fluorescence intensity in LRs is shown. LR were selected from the same position on the primary root. a.u., arbitrary units. Data are mean values (±SE) of three independent experiments. Different letters indicate a significant difference at P≤0.05 (Tukey test).
Figure 6.
miR393 early regulates redox components under salt.
Seven dpg WT and mir393ab seedlings were transferred onto liquid ATS medium supplemented with 100 mM NaCl. After 12 h of initial treatment (A) H2O2 accumulation, (B) APX activity and (C) CAT activity were measured. (D) Seven dpg mir393ab, tir1-1, tir1-1 35Spro:TIR-Myc 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).
Figure 7.
Modulation of plant development and acclimation to salinity are interconnected responses influenced by miR393 regulation node.
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.