Figure 1.
Expression pattern analysis of AGB1 in Arabidopsis and drought tolerance assay of agb1.
(A) Expression patterns of AGB1 were identified after treatments with 200 μM ABA and drought stress for 0, 0.5, 1, 2, 4, 6, 8, 12, and 24 h. The expression value of AGB1 at 0 h was normalized as 1; expression values at other time points were relative to the value at 0 h. Values represent means ± SD with three biological replicates. (B) Drought tolerance assay of WT (Col-0) and agb1-2 plants. WT and agb1-2 plants (before drought) were subjected to water stress by withholding water for 21 days (drought) and were then re-watered for 5 days (5 d after re-watering). (C) Survival rates of the WT and agb1-2 in (B) after drought stress. Data represent means ± SD (n = 60) from three independent experiments. Asterisks indicate significant differences (Student’s t test, **P<0.01) between WT and agb1-2. (D) Proline contents of WT and agb1-2 plants after dehydration treatments. For the proline assay, 14-day-old seedlings were subjected to drought treatment for 0, 1 and 3 h. Proline contents of WT and agb1-2 plants at the same time points were significantly different at 1 and 3 h. Data represent means ± SD from three independent experiments. Asterisks indicate significant differences (Student’s t test, **P<0.01) between WT and agb1-2. (E) and (F) Expression analysis of the proline biosynthesis genes ProDH (E) and P5CS2 (F) in WT and agb1-2 seedlings after drought treatment for 0, 0.5 and 1 h. All results are means ± SD for three independent experiments.
Figure 2.
AtMPK6 interacted with AGB1 in Y2H, Pull-down, BiFC and Co-IP assays.
(A) Interaction tests using yeast two-hybrid assays between AGB1 and AtMPK6. Yeast cells with AGB1 (AD-AGB1) and AtMPK6 (BD-MPK6) were placed in different liquid concentrations on control medium SD/-Trp/-Leu (SD/LT) and selection medium SD/-Trp/-Leu/-His/-Ade (SD/LTHA). For negative controls, pGBKT7 without insert (BD alone), pGADT7 without insert (AD alone), and the empty vectors AD and BD were used. Experiments were performed three times and a representative result is shown. (B) AGB1 interacted with AtMPK6 by BiFC assays in Arabidopsis protoplast. The recombinant constructs AGB1-YFPN (YFP N-terminal) and MPK6-YFPC (YFP C-terminal) were co-transformed into protoplast cells of Arabidopsis WT (Col-0). For negative controls, pSPYNE without insert AGB1 (YFPN) (iii) and pSPYCE without insert MPK6 (YFPC) (ii) were used. The combination of bZIP63-YFPN and bZIP63-YFPC was used as a positive control (iiii), Fluorescence was recorded 20 h after transformation. Experiments were performed 10 times and a representative result is shown. Bars = 40 μm in (i), (iii) and (iiii), and 20 μm in (ii). (C) Interaction assay using pull-down analysis. AGB1 and AtMPK6 fused with GST and His tags, respectively were mixed and passed through a glutathione column (binding GST tag). After elution with pull-down binding buffer, samples were separated by SDS-PAGE, and His-MPK6 was detected by immunoblotting with anti-His antibody. GST alone was used as the negative control. Experiments were performed three times and a representative result is shown. (D) Co-IP assay of AGB1 with AtMPK6. AGB1-Flag and MPK6-Myc or AGB1-Flag and empty pCAMBIA1300 vector (contained Myc-tags) were transiently co-transformed into Arabidopsis protoplast. After 16 h, the total Arabidopsis cell lysates were prepared for Co-IP with anti-Myc agarose. Then, anti-Myc immunoprecipitates were subjected to Western blot analysis with anti-Flag antibody (top). Meanwhile, the total cell lysates were also subjected to Western blot analysis with anti-Flag (middle), and anti-Myc (bottom, for MPK6-Myc expression) antibodies. Experiments were performed three times and a representative result is shown.
Figure 3.
Subcellular localization analysis of AtMPK6 and AtAGB1.
35S:AtMPK6-GFP was transiently expressed in protoplast cells of Arabidopsis strains WT (Col-0) and the agb1-2 mutant in (A) and (B). Subcellular localizations of AtMPK6-GFP were detected without ABA treatment (A) and with 10 μM ABA (B). Results were visualized by confocal microscopy. (C) Subcellular localization of AGB1. Each experiment was performed three times, and thirty cells were observed for each construct and a representative result is shown (Bars = 20 μm).
Figure 4.
Expression of AtVIP1 and AtMYB44 in WT and agb1-2 after ABA and drought treatments.
(A) and (B) Expression analysis of AtVIP1 and AtMYB44 in WT and agb1-2 plants after ABA (A) and drought (B) treatments for 0, 0.5, 1, 2, 4, 6, 8, 12, 16, and 24 h. Results are means ± SD with three biological replicates.
Figure 5.
Expression of stress-responsive and ABA-biosynthesis genes in WT and agb1-2 plants after drought treatment.
(A) Expression of RD29A, RAB18, and ERD10 in WT and agb1-2 after drought treatment for 0, 0.5, 1, 2, 4, 6, 8, 12, 16, and 24 h. (B) Expression of ABA biosynthesis genes ABA1 and ABA2 in WT and agb1-2 after drought treatment for 0, 0.5, 1, and 2 h. Results are means ± SD with three biological replicates.
Figure 6.
Overexpression of AtVIP1 inhibited seed germination and root length under ABA treatment.
Identically stored seeds were surface sterilized and washed extensively with water and plated on MS0 medium plates containing 3% Suc in the absence or presence of 2.5 μM ABA. Plates were kept at 4°C in darkness for 3 d and then transferred to growth chambers (16 h light/8 h darkness regime) at 22°C. (A) Seed germination analysis of WT and 35S:AtVIP1 transgenic plants containing OE-VIP1–4, -5 and -7 under normal conditions (MS0) and ABA treatment (MS0 plus 2.5 μM ABA). (B) and (C) Seed germination rates of different 35S:AtVIP1 transgenic lines under normal conditions (B), and 2.5 μM ABA treatment (C) at different time points. 35S:AtVIP1 transgenic lines displayed lower germination rates than WT. Values for each time point are means of three experiments, and each experiment comprised 60 plants. (D) Root growth WT and 35S:AtVIP1 transgenic plants under normal conditions (MS0) and 5 μM ABA treatment for 14 d. (E) Primary root lengths (cm) shown in (D) were measured 14 days after treatment. Values are means ± SD (n = 30) from three independent experiments. Asterisk indicates significant differences (Student’s t test,*P<0.05) between the Col-0 and 35S:AtVIP1 transgenic lines. (F) Relative expression analysis (fold change) of AtVIP1 in WT and transgenic lines. Expression of VIP1 in WT was normalized as 1. Results are means ± SD from three independent experiments, and asterisks indicate significant differences (Student’s t test,**P<0.01) between Col-0 and transgenic lines.
Figure 7.
Drought tolerance analysis of 35S:AtVIP1 transgenic plants.
(A) Growth of WT and 35S:AtVIP1 transgenic lines containing OE-VIP1–5 and -7 under normal conditions (MS0) and drought stress (MS0 plus 4% PEG). (B) Primary root lengths (cm) were measured at 14 days after PEG treatment. Values are means ± SD (n = 30) from three independent experiments and asterisks indicate significant differences (Student’s t test,*P<0.05 or **P<0.01) between WT and 35S::AtVIP1 transgenic plants. (C) Drought tolerance assay of the WT and 35S::AtVIP1 transgenic lines in soil. WT plants and 35S:AtVIP1 transgenic lines (21 days old, before drought treatment) were withheld from watering for 21 days and then re-watered for 3 days. Values under the figures are survival rates. Three independent experiments were conducted.
Figure 8.
A mode for AGB1 functions in ABA- and drought stress response through affecting AtMPK6-related pathway.
AGB1 negatively modulates ABA and drought response in Arabidopsis by down-regulating AtMPK6, AtVIP1 and AtMYB44 cascades and suppresses ABA biosynthesis and proline accumulation through regulating expression of ABA1, ABA2, ProDH and P5CS2. Positive effects are indicated by arrows and bars indicate repression.