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, 174 (4), 2515-2531

AtAIRP2 E3 Ligase Affects ABA and High-Salinity Responses by Stimulating Its ATP1/SDIRIP1 Substrate Turnover

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AtAIRP2 E3 Ligase Affects ABA and High-Salinity Responses by Stimulating Its ATP1/SDIRIP1 Substrate Turnover

Tae Rin Oh et al. Plant Physiol.

Abstract

AtAIRP2 is a cytosolic RING-type E3 ubiquitin ligase that positively regulates an abscisic acid (ABA) response in Arabidopsis (Arabidopsis thaliana). Yeast two-hybrid screening using AtAIRP2 as bait identified ATP1 (AtAIRP2 Target Protein1) as a substrate of AtAIRP2. ATP1 was found to be identical to SDIRIP1, which was reported recently to be a negative factor in ABA signaling and a target protein of the RING E3 ligase SDIR1. Accordingly, ATP1 was renamed ATP1/SDIRIP1. A specific interaction between AtAIRP2 and ATP1/SDIRIP1 and ubiquitination of ATP1/SDIRIP1 by AtAIRP2 were demonstrated in vitro and in planta. The turnover of ATP1/SDIRIP1 was regulated by AtAIRP2 in cell-free degradation and protoplast cotransfection assays. The ABA-mediated germination assay of 35S:ATP1/SDIRIP1-RNAi/atairp2 double mutant progeny revealed that ATP1/SDIRIP1 acts downstream of AtAIRP2. AtAIRP2 and SDIR1 reciprocally complemented the ABA- and salt-insensitive germination phenotypes of sdir1 and atairp2 mutants, respectively, indicating their combinatory roles in seed germination. Subcellular localization and bimolecular fluorescence complementation experiments in the presence of MG132, a 26S proteasome inhibitor, showed that AtAIRP2 and ATP1/SDIRIP1 were colocalized to the cytosolic spherical body, which lies in close proximity to the nucleus, in tobacco (Nicotiana benthamiana) leaf cells. The 26S proteasome subunits RPN12a and RPT1 and the molecular chaperones HSP70 and HSP101 were colocalized to these discrete punctae-like structures. These results raised the possibility that AtAIRP2 and ATP1/SDIRIP1 interact in the cytosolic spherical compartment. Collectively, our data suggest that the down-regulation of ATP1/SDIRIP1 by AtAIRP2 and SDIR1 RING E3 ubiquitin ligases is critical for ABA and high-salinity responses during germination in Arabidopsis.

Figures

Figure 1.
Figure 1.
AtAIRP2 interacts with ATP1/SDIRIP1. A, Yeast two-hybrid assay. AtAIRP2 and ATP1/SDIRIP1 were cloned into pGBKT7 and pGADT7, respectively. Yeast AH109 cells were cotransformed with a combination of the indicated plasmids. The yeast cells were plated onto SD/-Trp/-Leu/-His/-Ade medium and allowed to grow for 4 d at 30°C. p53 + T-antigen was used as a positive control, and lambda + T-antigen was used as a negative control. aa, Amino acids. B, In vitro pull-down assay. Bacterially expressed 6×His-3×HA-ATP1 and MBP-AtAIRP2 fusion proteins were coincubated in the presence of an amylose gel matrix. The resin-bound proteins were eluted with 2× SDS sample buffer and subjected to immunoblot analysis using anti-HA and anti-MBP antibodies. C, In vivo co-IP assay. The 35S:ATP1/SDIRIP1-3×Myc and 35S:2×Flag-AtAIRP2 constructs were infiltrated into tobacco leaves. Leaf crude extract (1 mg of protein) was immunoprecipitated with anti-Flag affinity gel matrix. The bound proteins were eluted, separated by SDS-PAGE, and detected with anti-Flag or anti-Myc antibody. D and E, In vitro target ubiquitination assays. Recombinant 6×His-ATP1/SDIRIP1-6×Myc protein was incubated in the presence or absence of E1, E2, MBP-AtAIRP2, or MBP-AtAIRP2H163A for 1 h and subjected to immunoblotting using anti-Myc (D) and anti-Ub (E) antibodies. The asterisk indicates the shifted high-molecular-mass band. The vertical dashed line indicates a ubiquitinated smear ladder.
Figure 2.
Figure 2.
The turnover of ATP1/SDIRIP1 was regulated by AtAIRP2 in a 26S proteasome-dependent manner. A and B, In vitro cell-free degradation assay of ATP1/SDIRIP1. Bacterially expressed 6×His-ATP1/SDIRIP1-6×Myc and 6×His-RGA1-2×Flag were incubated with leaf crude extracts prepared from 14-d-old wild-type plants treated with mock or 50 µm ABA (6 h) for the indicated time periods (A). The recombinant ATP1/SDIRIP1 and RGA1 were incubated with leaf crude extracts prepared from 14-d-old wild-type (WT), 35S:AtAIRP2-sGFP-overexpressing, or atairp2-1 mutant plants in the absence (0, 0.5, 1, and 2 h) or presence (2 h) of 40 µm MG132 (B). The protein levels were measured by immunoblot analysis using anti-Myc and anti-Flag antibodies. DSred2 and Rubisco large subunit were used as loading controls. The time-dependent degradation patterns of each protein were quantified using ImageJ software. Results are presented as means ± sd (*, P < 0.05 and **, P < 0.01, Student’s t test) of three independent biological replicates. C, Apparent half-lives of 6×His-ATP1/SDIRIP1-6×Myc in the cell-free crude extracts. The graph shows the time-dependent decrease in the relative amount of 6×His-ATP1/SDIRIP1-6×Myc in the cell-free degradation assay. The levels of proteins were quantified by calculation of band intensities using ImageJ software. D and E, Protoplast cotransfection assay. The 35S:AtAIRP2-sGFP, 35S:sGFP, 35S:ATP1/SDIRIP1-3×Myc, and 35S:DSred2 constructs (D) or the 35S:AtAIRP2-sGFP, 35S:ATP1/SDIRIP1-3×Myc, and 35S:DSred2 constructs (E) were introduced into Arabidopsis leaf protoplasts by the polyethylene glycol-mediated transformation method. The transformed protoplasts were incubated in washing and incubation solution (4 mm MES, pH 5.7, 0.5 m mannitol, and 20 mm KCl) at 22°C under conditions of continuous light. After 16 h of incubation, protoplasts were incubated with or without 40 µm MG132 for 2 h (E). Total proteins were extracted from the protoplasts and subjected to immunoblotting with anti-Myc, anti-GFP, and anti-DSred2 antibodies. Results are presented as means ± sd (**, P < 0.01, Student’s t test) of three independent biological replicates.
Figure 3.
Figure 3.
AtAIRP2 is epistatic to ATP1/SDIRIP1 in ABA-mediated seed germination. A, Germination assays of wild-type (WT), atairp2-1, 35S:ATP1/SDIRIP1-RNAi (independent lines 1 and 2), and 35S:ATP1/SDIRIP1-RNAi/atairp2-1 plants in response to ABA. Sterilized seeds were imbibed in water for 2 d at 4°C and incubated on Murashige and Skoog (MS) medium in the presence of different concentrations (0, 0.5, and 1 μm) of ABA at 22°C under a 16-h-light/8-h-dark photoperiod. Bars = 0.5 cm. B and C, Germination percentages were determined in terms of radicle emergence 3 d after germination (B) and cotyledon greening 8 d after germination (C). Results are presented as means ± sd (**, P < 0.01, Student’s t test) of three independent biological replicates (n = 25).
Figure 4.
Figure 4.
Combinatorial down-regulation of ATP1/SDIRIP1 by AtAIRP2 and SDIR1 RING E3 Ub ligases. A and C, The indicated combinations of 35S:ATP1/SDIRIP1-3×Myc, 35S:AtAIRP2-sGFP, 35S:2×Flag-SDIR1, and 35S:DSred2 constructs were transformed into the protoplasts prepared from wild-type (WT; A and C), atairp2-1 (A), or sdir1-1 (C) leaves and incubated in washing and incubation solution for 16 h at 22°C under conditions of continuous light. The protein levels were examined by immunoblot analysis with anti-Myc, anti-GFP, anti-Flag, and anti-DSred2 antibodies. B and D, Protein levels were quantified by calculation of band intensities using ImageJ software. DSred2 and Rubisco large subunit were used as loading controls. Bars represent means ± se (*, P < 0.05 and **, P < 0.01, Student’s t test) of three independent biological replicates.
Figure 5.
Figure 5.
Reciprocal complementation assays of AtAIRP2 and SDIR1 RING E3 Ub ligases in ABA-mediated seed germination. A and B, RT-PCR (A) and immunoblot (B) analyses. 35S:AtAIRP2-sGFP and 35S:2×Flag-SDIR1 were transformed into sdir1-1 and atairp2-1 mutants, respectively. Expression levels of SDIR1 and AtAIRP2 were examined in atairp2-1/35S:2×Flag-SDIR1 (lines 1 and 5) and sdir1-1/35S:AtARIP2 (lines 4 and 11) complementation T3 transgenic plants, respectively, by RT-PCR (A) and immunoblotting (B). AtUBC10 and Rubisco large subunit were used as loading controls. C, Seed germination assays of atairp2-1/35S:2×Flag-SDIR1 and sdir1-1/35S:AtARIP2 complementation T3 transgenic plants in response to ABA. After imbibition in water for 2 d at 4°C, wild-type (WT), atairp2-1 and sdir1-1 mutant, and atairp2-1/35S:2xFlag-SDIR1 and sdir1-1/35S:AtAIRP2-sGFP complementation seeds were incubated on MS medium in the presence of different concentrations (0, 0.5, and 1 μm) of ABA at 22°C under a 16-h-light/8-h-dark photoperiod. Bars = 0.5 cm. D and E, Germination percentages were measured with respect to radicle emergence 3 d after germination (D) and cotyledon greening 8 d after germination (E). Results are presented as means ± sd (*, P < 0.05 and **, P < 0.01, Student’s t test) of three independent biological replicates (n = 25).
Figure 6.
Figure 6.
High-salinity responses of atairp2-1/35S:2×Flag-SDIR1 and sdir1-1/35S:AtARIP2 complementation T3 transgenic plants at the germination stage. A, Seed germination assays of atairp2-1/35S:2×Flag-SDIR1 and sdir1-1/35S:AtAIRP2-sGFP complementation lines under high-salinity conditions. Wild-type (WT), atairp2-1 and sdir1-1 mutant, and atairp2-1/35S:2×Flag-SDIR1 and sdir1-1/35S:AtAIRP2-sGFP complementation seeds were treated with different concentrations of NaCl (0, 100, and 150 mm) at 22°C under a 16-h-light/8-h-dark photoperiod. Bars = 0.5 cm. B and C, Germination percentages were evaluated in terms of radicle emergence 3 d after germination (B) and cotyledon greening 8 d after germination (C). Data represent means ± sd (*, P < 0.05 and **, P < 0.01, Student’s t test) of three biological replicates (n = 25). D, Reciprocal qRT-PCR analysis of the ABA-responsive transcription factor ABI5. The ABI5 transcript levels were examined in wild-type, atairp2 and sdir1 mutant, and atairp2-1/35S:2×Flag-SDIR1 and sdir1-1/35S:AtARIP2 complementation T3 transgenic plants by qRT-PCR. Arabidopsis glycerealdehyde-3-phosphate dehydrogenase was used as an internal control.
Figure 7.
Figure 7.
Subcellular localization and BiFC assays of AtAIRP2 and ATP1/SDIRIP1. A, Subcellular localization of AtAIRP2 and ATP1/SDIRIP1 in tobacco leaf epidermal cells. 35S:AtAIRP2-sGFP and 35S:ATP1/SDIRIP1-mRFP were introduced into tobacco epidermal cells by an A. tumefaciens-mediated transient expression method. After 2 d of incubation at 25°C under a 16-h-light/8-h-dark photoperiod, tobacco cells were treated with or without 20 μm MG132 for 4 h. The fluorescent signals from AtAIRP2-sGFP and ATP1/SDIRIP1-mRFP were detected via confocal microscopy. Arrowheads indicate cytosolic spherical bodies, in which colocalized signals of AtAIRP2-sGFP and ATP1/SDIRIP1-mRFP were detected. Bars = 10 μm. B, BiFC analysis of the in planta interaction between AtAIRP2 and ATP1/SDIRIP1. The full-length coding sequences of ATP1/SDIRIP1 and AtAIRP2 were fused to the N-terminal (nYFP) and C-terminal (cYFP) regions of YFP, respectively. The ATP1/SDIRIP1-nYFP + cYFP, AtAIRP2-cYFP + nYFP, and ATP1SDIRIP1-nYFP + AtAIRP2-cYFP proteins were coexpressed in tobacco leaf cells along with NLS-mRFP, a nuclear marker protein. After 2 d of incubation, tobacco cells were treated with 20 μm MG132 for 4 h. Reconstituted fluorescent signals were visualized by confocal microscopy. The arrowheads indicate BiFC signals from the interaction between ATP1/SDIRIP1-nYFP and AtAIRP2-cYFP, and the asterisks indicate nuclei. Bars = 10 μm.
Figure 8.
Figure 8.
Colocalization of the 26S proteasome subunits (RPN12a and RPT1) and molecular chaperones (HSP70 and HSP101) to the cytosolic spherical compartment. A, ATP1/SDIRIP1-nYFP + AtAIRP2-cYFP proteins were coexpressed with RPN12a-mRFP (left) or RPT1-mRFP (right) in tobacco leaf cells in the presence of 20 μm MG132. The fluorescent signals from YFP and mRFP were detected using confocal microscopy. The arrowheads indicate cytosolic spherical bodies, in which AtAIRP2, ATP1/SDIRIP1, RPN12a, and RPT1 proteins are colocalized, and the asterisks indicate nuclei. Bars = 10 μm. B, ATP1/SDIRIP1-nYFP + AtAIRP2-cYFP proteins were coexpressed with mRFP-HSP70 (left) or mRFP-HSP101 (right) in tobacco leaf cells in the presence of 20 μm MG132. Bars = 10 μm.
Figure 9.
Figure 9.
Working model of AtAIRP2 and SDIR RING E3 Ub ligases in ABA and high-salinity responses during seed germination. In response to ABA and high-salt stress, ATP1/SDIRIP1 is ubiquitinated by AtAIRP2 in the cytosol and then rapidly degraded by the 26S proteasome complex before ATP1/SDIRIP1 enters the chloroplasts and/or nucleus. In addition, ATP1/SDIRIP1 is ubiquitinated by SDIR1 on the cytosolic side of the ER membrane (Zhang et al., 2015). The proteasome-dependent down-regulation of ATP1/SDIRIP1 by the combinatorial actions of two RING-type E3 Ub ligases results in the increased response to ABA and high salinity in the germination stage. The possible role of the cytosolic spherical body in the interaction of AtAIRP2 and ATP1/SDIRIP1 remains to be determined.

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