The Pepper RING-Type E3 Ligase CaAIRF1 Regulates ABA and Drought Signaling via CaADIP1 Protein Phosphatase Degradation

Abstract

Ubiquitin-mediated protein modification occurs at multiple steps of abscisic acid (ABA) signaling. Here, we sought proteins responsible for degradation of the pepper (Capsicum annuum) type 2C protein phosphatase CaADIP1 via the 26S proteasome system. We showed that the RING-type E3 ligase CaAIRF1 (Capsicum annuum ADIP1 Interacting RING Finger Protein 1) interacts with and ubiquitinates CaADIP1. CaADIP1 degradation was slower in crude proteins from CaAIRF1-silenced peppers than in those from control plants. CaAIRF1-silenced pepper plants displayed reduced ABA sensitivity and decreased drought tolerance characterized by delayed stomatal closure and suppressed induction of ABA- and drought-responsive marker genes. In contrast, CaAIRF1-overexpressing Arabidopsis (Arabidopsis thaliana) plants exhibited ABA-hypersensitive and drought-tolerant phenotypes. Moreover, in these plants, CaADIP1-induced ABA hyposensitivity was strongly suppressed by CaAIRF1 overexpression. Our findings highlight a potential new route for fine-tune regulation of ABA signaling in pepper via CaAIRF1 and CaADIP1.

Figure 1.

ABA-promoted degradation of the CaADIP1 protein in vitro. A, Polyubiquitination of the CaADIP1 protein. Leaves of tobacco plants harboring Pro-35S:CaADIP1-GFP were harvested 6 h after treatment with 100 μm ABA. At 12 h before sample harvesting, 50 μm MG132 was infiltrated into the leaves. Protein extracts were immunoprecipitated using a GFP-trap, followed by immunoblot analysis. Polyubiquitinated CaADIP1 protein was detected using anti-GFP (top) and anti-Ub (bottom). Coomassie blue staining (CBB) staining indicates equal loading of protein extract. fGFP, fragmented GFP. B, ABA-promoted in vivo degradation of CaADIP1. Leaves of tobacco plants harboring Pro-35S:CaADIP1-HA were infiltrated with 50 μm CHX 2 d after agroinfiltration and harvested at the indicated time points after treatment with 100 μm ABA. Immunoblot analysis was performed using anti-HA antibody, and the relative intensities of the CaADIP1-HA fusion proteins were measured with Image J 1.46r (http://imagej.nih.gov/ij) software. CBB staining indicates equal loading of total protein. C, Cell-free degradation of the CaADIP1 protein. The GST-tagged CaADIP1 (500 ng) protein was incubated for the indicated periods with crude extracts prepared from the leaves of ABA-treated 4-week-old pepper plants. Immunoblot analysis was performed using anti-GST antibody (top). CBB staining indicates equal loading of crude extract (bottom). The relative intensities of the GST-CaADIP1 fusion proteins were measured. All data represent the mean ± sd of three independent experiments; asterisks indicate significant differences (Student’s t test; P < 0.05).

Figure 2.

Interaction of CaADIP1 with CaAIRF1. A, Yeast two-hybrid assay of interactions between CaADIP1 and CaAIRF1. Interaction was indicated by growth on selection medium (SC-adenine-His-Leu-Trp; left); growth on SC-LW was used as a control (right). B, Co-IP of CaADIP1-HA and CaAIRF1^H864Y/C868S-GFP. For efficient interaction with CaADIP1, CaAIRF1^H864Y/C868S protein showing loss of E3 ligase activity was used instead of intact CaAIRF1. CaADIP1-HA was coexpressed with CaAIRF1^H864Y/C868S-GFP or GFP alone (negative control) in the leaves of tobacco. Immunoblot analysis was performed using anti-HA and anti-GFP antibodies. C, BiFC assay of interactions between CaADIP1 and CaAIRF1. CaADIP1-VYNE was coexpressed with CaAIRF1-CYCE in the leaves of tobacco. For the negative control, CaADIP1-VYNE and CaAIRF1-CYCE were coexpressed with CYCE and VYNE, respectively. Scale bar = 10 μm. D, Domain organization of deduced amino acids in the CaAIRF1 protein. The conserved domain was analyzed using the SMART Web site (http://smart.embl-heidelberg.de) and framed in the green (coiled-coil region) and pink (RING domain) boxes (top). Multiple alignment analysis of the RING domains of CaAIRF1 and its homologous proteins was performed using ClustalW2 (bottom). Amino acid residues of RING domains are shaded according to the percentage identity in ClustalW2; key residues are boxed in red (see also Supplemental Fig. S1). E, Organ-specific expression of CaAIRF1 in pepper plants. The pepper Actin1 (CaACT1) gene was used as an internal control. F, Subcellular localization of CaAIRF1 using transient expression of the GFP fusion protein in tobacco. White bar = 10 μm.

Figure 3.

ABA-promoted expression of the pCaAIRF1::EGFP fusion gene. A, RT-PCR analysis of CaAIRF1 expression. The expression pattern of CaAIRF1 was analyzed in the leaves of pepper plants treated with ABA (100 μm), H₂O₂ (100 μm), drought, or NaCl (200 mm). The pepper Actin1 (CaACT1) gene was used as an internal control. B, Schematic representation of the transfer DNA (T-DNA) region of the pHGWFS7 vector containing a 1,504-bp upstream region of CaAIRF1. The colored boxes indicate cis-regulatory elements, associated with ABA signaling, in the CaAIRF1 promoter sequence. HRT, hygromycin phosphotransferase; LB, left border; RB, right border. C and D, Induction of GFP expression in response to ABA. Leaves were harvested from nontransgenic (NT) and transgenic tobacco plants 24 h after treatment with 50 μm ABA. Immunoblot (C) and qRT-PCR (D) analyses were performed using an tobacco leaf harboring the ProCaAIRF1::EGFP fusion gene. The polyclonal anti-GFP antibody was used. For qRT-PCR, the expression level of the GFP gene was normalized with that of CaACT1. Data represent the mean ± sd of three independent experiments; asterisks indicate significant differences (Student’s t test; P < 0.05). E and F, GFP signals from the leaves of tobacco (E) and pepper (F) plants. After agroinfiltration, leaves were treated with 50 μm ABA for 24 h and subjected to microscopic analysis. White bar = 50 μm (E) and 10 μm (F).

Figure 4.

CaAIRF1-mediated degradation of CaADIP1. A, In vitro E3 Ub ligase activity of the CaAIRF1∆1-573 protein. MBP-tagged recombinant CaAIRF1∆1-573 and double-amino acid substitution mutant CaAIRF1∆1-573^H864Y/C868S proteins were assayed for E3 activity in the presence or absence of Arabidopsis E1, E2, Ub, and ATP. Immunoblot analyses were performed using anti-MBP (top) and anti-Ub (bottom) antibodies. B, In vitro ubiquitination of CaADIP1 by CaAIRF1. MBP-CaAIRF1∆1-573 and MBP-CaAIRF1∆1-573^H864Y/C868S were incubated for 2 h with GST-CaADIP1 in the presence or absence of E1, E2, and Ub. Immunoblot analyses were performed using anti-GST antibody. C and D, CaAIRF1-mediated polyubiquitination of CaADIP1 in vivo. In the tobacco leaves, Pro-35S:CaADIP1-HA was coexpressed with Pro-35S:GFP (#1), Pro-35S:CaAIRF1-GFP (#2), or Pro-35S:CaAIRF1^H864Y/C868S-GFP (#3). At 12 h before sample harvesting, 50 μm MG132 was infiltrated into the leaves. C, Total protein extracts were prepared 3 d after agroinfiltration and subjected to immunoblot analysis using anti-HA and anti-GFP antibodies. D, Using the UbiQapture-Q matrix, all ubiquitinated proteins were isolated from the same protein samples. Immunoblot analysis was performed using anti-GFP (right), anti-HA (middle), and anti-Ub (right) antibodies.

Figure 5.

CaAIRF1-mediated degradation of CaADIP1 in a 26S proteasome-dependent manner. A, In vivo assay of CaAIRF1-mediated degradation of CaADIP1. Through agroinfiltration, Pro-35S:CaADIP1-HA was coexpressed with Pro-35S:GFP, Pro-35S:CaAIRF1-GFP, or Pro-35S:CaAIRF1^H864Y/C868S-GFP in the leaves of tobacco plants. At 6 h before sample harvesting, 50 μm CHX and 50 μm MG132 were infiltrated into the leaves. Protein extracts were subjected to immunoblot analysis. The relative intensities of the CaADIP1-HA fusion proteins were measured using Image J 1.46r (http://imagej.nih.gov/ij) software (bottom). CBB staining indicates equal loading of crude extract. B, Expression level of CaAIRF1 in the leaves of CaAIRF1-silenced pepper plants. TRV2:CaAIRF1 and TRV2:00 control pepper plants were treated with 10 μm ABA; after 24 h, leaves of each line were harvested. The relative expression level of CaAIRF1 was examined using qRT-PCR analysis and normalized to that of CaACT1 as an internal control gene. Data represent the mean ± sem of three independent experiments. C, Cell-free degradation assay for CaADIP1. The GST-CaADIP1 (500 ng) protein was incubated for 30 min with crude extracts prepared from the leaves of 4-week-old TRV:CaAIRF1 pepper plants. Immunoblot analysis was performed using anti-GST antibody (top). CBB staining indicates equal loading of crude extract. The relative intensities of the GST-CaADIP1 fusion proteins were measured using Image J 1.46r (http://imagej.nih.gov/ij) software (bottom). Data represent the mean ± sd of three independent experiments; asterisks indicate significant differences (Student’s t test; P < 0.05).

Figure 6.

Suppression of ABA-mediated stomatal closing and ABA-responsive gene expression in CaAIRF1-silenced pepper leaves. A and B, Representative thermographic images of CaAIRF1-silenced pepper plants 6 h after treatment with 50 μm ABA (A); the mean leaf temperature was measured using 10 plants of each line (B). Data represent the mean ± sd of three independent experiments. C and D, Stomatal apertures in control and CaAIRF1-silenced pepper plants treated with ABA. Leaf peels harvested from 4-week-old plants of each line were incubated for 2 h in SOS buffer containing 20 μm ABA. Representative images were taken (C) and the stomatal apertures were measured under the microscope (D). Data represent the mean ± se of three independent experiments. E, RT-PCR analysis of ABA-responsive gene expression in the leaves of TRV2:00 and TRV2:CaAIRF1 pepper plants. Four-week-old plants of each line were treated with 10 μm ABA; after 24 h, leaves of each line were harvested. The relative expression level (∆∆CT) of each gene was normalized to that of CaACT1 as an internal control gene. Data represent the mean ± se of three independent experiments. F and G, Floating leaf assay of TRV2:00 and TRV2:CaAIRF1 pepper plants. The first and second fully expanded leaves of each plant line (4 weeks old) were floated in SOS buffer containing 50 μm ABA. After 6 d, representative images were taken (F) and the relative chlorophyll content of each leaf was measured (G). Data represent the mean ± sd of three independent experiments. Asterisks indicate significant differences (Student’s t test; P < 0.05). For all experiments, the first and second leaves of pepper plants were used.

Figure 7.

Increased susceptibility of CaAIRF1-silenced pepper plants to dehydration stress. A and B, Representative thermographic images of detached leaves from TRV2:00 and TRV2:CaAIRF1 pepper plants (A); the mean leaf temperature was measured in the first and second leaves of each line (n = 10) (B). Data represent the mean ± sd of three independent experiments. C, Water loss from the leaves of TRV2:00 and TRV2:CaAIRF1 pepper plants at various time points after detachment of leaves. The leaf fresh weights of each line were measured 8 h after detachment of leaves. Data represent the mean ± se of three independent experiments, each evaluating 16 plants. D, Dehydration sensitivity of CaAIRF1-silenced pepper plants. Four-week-old TRV2:CaAIRF1 and TRV2:00 pepper plants were subjected to dehydration stress by withholding watering for 15 d. Representative images were taken before (left) and after (middle) dehydration stress and 3 d after rewatering (right). The survival rate was measured 3 d after rewatering. Data represent the mean ± sd of three independent experiments. E, RT-PCR analysis of ABA-responsive gene expression in dehydrated leaves of TRV2:00 and TRV2:CaAIRF1 pepper plants. Four-week-old plants of each line were subjected to dehydration stress by removal of their roots. The relative expression level (∆∆CT) of each gene was normalized to that of CaACT1 as an internal control gene. Data represent the mean ± se of three independent experiments. Asterisks indicate significant differences (Student’s t test; P < 0.05).

Figure 8.

Enhanced tolerance of Pro-35S:CaAIRF1 transgenic Arabidopsis plants to dehydration stress. A and B, Representative thermographic images of Pro-35S:CaAIRF1 transgenic Arabidopsis lines and wild-type (WT) plants 6 h after treatment with 50 μm ABA (A); the mean leaf temperatures of the three largest leaves were measured using 10 plants of each line (B). Data represent the mean ± sd of three independent experiments. C and D, Stomatal apertures in Pro-35S:CaAIRF1 transgenic Arabidopsis lines and wild-type plants treated with ABA. Leaf peels harvested from 3-week-old plants of each line were incubated for 2 h in SOS buffer containing 10 μm ABA. The stomatal apertures were measured under the microscope (C) and representative images were taken (D). Data represent the mean ± se of three independent experiments. E, Transpirational water loss from the leaves of Pro-35S:CaAIRF1 transgenic lines and wild-type plants. The fresh weights of each line were measured 7 h after detachment of leaves. Data represent the mean ± se of three independent experiments, each evaluating 16 plants. F, Dehydration sensitivity of Pro-35S:CaAIRF1 transgenic plants. Three-week-old wild-type and transgenic plants were subjected to dehydration stress by withholding watering for 12 d. Representative images were taken, and the percentages of plants that survived were measured after rehydration for 3 d. Data represent the mean ± se of three independent experiments. G, Expression analysis of dehydration-responsive genes in the leaves of Pro-35S:CaAIRF1 transgenic lines and wild-type plants. The relative expression level (∆∆CT) of each gene was normalized to that of Actin8 as an internal control gene. Data represent the mean ± se of three independent experiments. Different letters indicate significant differences (ANOVA; P < 0.05).

Figure 9.

CaAIRF1-mediated inhibition of CaADIP1 function during germination and seedling development. A, Cell-free degradation assay for CaADIP1. The GST-CaADIP1 protein (500 ng) was incubated for the indicated periods with crude extracts prepared from the leaves of 4-week-old plants of the Pro-35S:CaAIRF1 transgenic line #6 in the presence or absence of MG132. Immunoblot analysis was performed using anti-GST antibody (top). CBB staining indicates equal loading of crude extract. The relative intensities of the GST-CaADIP1 fusion proteins were measured using Image J 1.46r (http://imagej.nih.gov/ij) software (bottom). Data represent the mean ± sd of three independent experiments; asterisks indicate significant differences (Student’s t test; P < 0.05). B, Germination rates of transgenic lines and wild-type (WT) plants on 0.5× MS medium supplemented with various concentrations of ABA. The numbers of seeds with emerged radicles were counted 2 d and 4 d after plating (DAP). Data represent the mean ± sd of three independent experiments, each evaluating 50 seeds. C, Seedling growth of transgenic lines and wild-type plants exposed to ABA. The seedlings were grown vertically in 0.5× MS containing 0.75 μm ABA. After 7 d, representative images were taken. D, Rate of cotyledon greening of transgenic lines and wild-type plants exposed to ABA. The numbers of seedlings in each line with expanded cotyledons were counted 7 d after plating. Data represent the mean ± sd of three independent experiments, each evaluating 45 seeds. Different letters indicate significant differences (ANOVA; P < 0.05).

Figure 10.

Schematic representation of the functional role of CaAIRF1 in the ABA-signaling pathway that mediates drought stress tolerance in pepper plants. We proposed that CaAIRF1 plays a positive role in ABA signaling via regulation of CaADIP1 protein stability and CaADIP1 transcript accumulation. This leads to alteration of downstream responses, including ABA-inducible gene expression and stomatal closure, and consequently controls plant tolerance to drought stress. Dashed lines indicate direct or indirect actions of CaAIRF1 and CaADIP1. Arrows indicate promotion actions; lines with end bar indicate inhibitory actions.