The Arabidopsis RING E3 ubiquitin ligase AtAIRP2 plays combinatory roles with AtAIRP1 in abscisic acid-mediated drought stress responses

Abstract

The ubiquitin (Ub)-26S proteasome pathway is implicated in various cellular processes in higher plants. AtAIRP1, a C3H2C3-type RING (for Really Interesting New Gene) E3 Ub ligase, is a positive regulator in the Arabidopsis (Arabidopsis thaliana) abscisic acid (ABA)-dependent drought response. Here, the AtAIRP2 (for Arabidopsis ABA-insensitive RING protein 2) gene was identified and characterized. AtAIRP2 encodes a cytosolic C3HC4-type RING E3 Ub ligase whose expression was markedly induced by ABA and dehydration stress. Thus, AtAIRP2 belongs to a different RING subclass than AtAIRP1 with a limited sequence identity. AtAIRP2-overexpressing transgenic (35S:AtAIRP2-sGFP) and atairp2 loss-of-function mutant plants exhibited hypersensitive and hyposensitive phenotypes, respectively, to ABA in terms of seed germination, root growth, and stomatal movement. 35S:AtAIRP2-sGFP plants were highly tolerant to severe drought stress, and atairp2 alleles were more susceptible to water stress than were wild-type plants. Higher levels of drought-induced hydrogen peroxide production were detected in 35S:AtAIRP2-sGFP as compared with atairp2 plants. ABA-inducible drought-related genes were up-regulated in 35S:AtAIRP2-sGFP and down-regulated in atairp2 progeny. The positive effects of AtAIRP2 on ABA-induced stress genes were dependent on SNF1-related protein kinases, key components of the ABA signaling pathway. Therefore, AtAIRP2 is involved in positive regulation of ABA-dependent drought stress responses. To address the functional relationship between AtAIRP1 and AtAIRP2, FLAG-AtAIRP1 and AtAIRP2-sGFP genes were ectopically expressed in atairp2-2 and atairp1 plants, respectively. Constitutive expression of FLAG-AtAIRP1 and AtAIRP2-sGFP in atairp2-2 and atairp1 plants, respectively, reciprocally rescued the loss-of-function ABA-insensitive phenotypes during germination. Additionally, atairp1/35S:AtAIRP2-sGFP and atairp2-2/35S:FLAG-AtAIRP1 complementation lines were more tolerant to dehydration stress relative to atairp1 and atairp2-2 single knockout plants. Overall, these results suggest that AtAIRP2 plays combinatory roles with AtAIRP1 in Arabidopsis ABA-mediated drought stress responses.

Figure 1.

Identification of atairp2 loss-of-function mutants, sequence analysis of the AtAIRP2 gene, and construction of AtAIRP2 overexpressors. A, Schematic representation of the atairp2-1 (SAIL_686_G08) and atairp2-2 (Salk_005082) alleles with T-DNA insertions. Gray bars indicate coding regions, black bars indicate the 5′ and 3′ untranslated regions, and solid lines represent introns of the AtAIRP2 gene (GenBank accession no. NM_120230). T-DNA insertions are indicated by triangles. T-DNA-specific (LB1 and LB2) and gene-specific (FW1, FW2, FW3, RV1, RV2, and RV3) primers used in genotyping PCR and RT-PCR are indicated with arrows. B, Genotyping PCR of the two atairp2 T-DNA insertion mutant alleles (atairp2-1 and atairp2). Gene-specific and T-DNA-specific primer sets used for genomic PCRs are indicated on the right. WT, Wild type. C, Expression levels of AtAIRP2 transcripts in wild-type and atairp2 mutant plants. Gene-specific primer sets for RT-PCR are indicated on the right. Constitutively expressed UBC10 (for E2 ubiquitin-conjugating enzyme) mRNA was used as a loading control. Primer sequences are listed in Supplemental Table S1. D, Schematic structure of the full-length AtAIRP2 cDNA clone and its deduced protein. The gray bar indicates the coding region, and solid lines represent the 5′ and 3′ untranslated regions. The C-terminal C3HC4-type RING domain is indicated by the black bar. E, Phylogenetic analysis of the seven AtAIRP2 homologs from Arabidopsis (At5g58787 and At3g47160), rice (GenBank accession no. NP_001060539), poplar (XP_002309135), grape (XP_002280008), and sorghum (XP_002447334). F, Amino acid sequence alignment of the RING motifs of AtAIRP2 and other C3HC4-type RING proteins. Potential Zn²⁺-interacting amino acid residues (C-X₂-C-X₁₁-C-X₁-H-X₂-C-X₂-C-X₁₀-C-X₂-C) are indicated. Amino acid residues identical in all seven RING domains are shown in black, and those conserved in at least four of the seven sequences are shaded. G, Real-time qRT-PCR analysis of the wild type and AtAIRP2 overexpressors. Expression levels of AtAIRP2 transcripts in wild-type and T3 35S:AtAIRP2-sGFP transgenic (independent lines 10 and 19) plants were determined by real-time qRT-PCR using gene-specific primer sets. UBC10 mRNA levels were used as a loading control. H, Immunoblot analysis of wild-type and AtAIRP2-sGFP (lines 10 and 19) plants. Expression levels of the AtAIRP2-sGFP fusion protein were determined using an anti-GFP antibody. Rubisco large subunit (RbcL) was used as a loading control.

Figure 2.

Expression profiles of AtAIRP2 in response to ABA and different abiotic stress conditions. A, Light-grown, 10-d-old Arabidopsis seedlings were treated with 100 μm ABA (1.5–3 h), drought (1–2 h), high salinity (300 mm NaCl for 1.5–3 h), or cold (4°C for 12–24 h). Total RNA was isolated from the treated tissues and used for RT-PCR. The RAB18 and RD29A genes were positive controls for ABA and abiotic stress responses, respectively. UBC10 was used as a loading control. B to D, AtAIRP2 promoter activity. AtAIRP2-promoter:GUS transgenic T3 plants were incubated with 5-bromo-4-chloro-3-indolyl-β-glucuronic acid for 12 h. AtAIRP2 promoter activity was visualized by GUS-specific staining. B, Histochemical localization of GUS activity in young seedlings (72 h after imbibition, 1 d after germination, 2 d after germination, and 4-d-old seedlings). Arrows indicate GUS signals. Bars = 0.25 cm. C, GUS-specific staining patterns in 10-d-old seedlings in response to ABA, drought, salt, and cold treatments. GUS signals were markedly induced in guard cells in rosette leaves and roots. Bar lengths are indicated to the right. D, GUS activity in mature plants. GUS signals were detected in anthers (flower buds), upper region of stigma (2–3 d after flowering [DAF]), and siliques (6–16 d after flowering). Bars = 0.25 cm. [See online article for color version of this figure.]

Figure 3.

AtAIRP2 is a cytosolic RING E3 Ub ligase. A, In vitro E3 Ub ligase assay. Left panel, bacterially expressed MBP-AtAIRP2 was incubated with ATP in the presence or absence of Ub, Arabidopsis E1 (His-UBA1), and Arabidopsis E2 (His-UBC8) at 30°C for 2 h. Reaction mixtures were separated by SDS-PAGE and subjected to immunoblot analysis using either anti-MBP antibody or anti-Ub antibody. Right panel, MBP-AtAIRP2 and single-amino acid substitution mutant MBP-AtAIRP1^H163A were incubated at 30°C for 2 h in the presence of ATP, Ub, E1, and E2. Ubiquitinated proteins were detected by either anti-MBP or anti-Ub antibody. WT, Wild type. B, Cytosolic localization of AtAIRP2. 35S:sGFP, 35S:AtAIRP2-sGFP, 35S:AtAIRP1-sGFP, and 35S:AREB1-sGFP gene constructs were transformed into onion epidermal cells using particle bombardment. Localization of the expressed proteins was visualized by fluorescence microscopy (dark and bright fields) in both unplasmolyzed and plasmolyzed onion cells. Arabidopsis AREB1 and RING E3 Ub ligase AtAIRP1 were used as specificity controls for nuclear and cytosolic proteins, respectively. DAPI, 4′,6-Diamino-phenylindole. Bars = 100 μm. [See online article for color version of this figure.]

Figure 4.

Germination rates of wild-type, atairp2, and 35S:AtAIRP2-sGFP plants in response to ABA and NaCl. A, ABA sensitivity of the wild type (WT), two atairp2 mutant alleles (atairp2-1 and atairp2-2), and AtAIRP2 overexpressors (transgenic lines 10 and 19) during the germination stage. Sterilized seeds were imbibed in water for 2 d at 4°C and incubated on MS medium in the presence of different concentrations of ABA (0, 0.2, 0.4, and 0.8 μm) at 22°C under a 16-h-light/8-h-dark photoperiod. Germination percentages were determined in terms of radical emergence 3 d after germination and cotyledon greening 7 d after germination. sd values were determined from four biological replicates (n > 36). Bars = 0.5 cm. B, NaCl sensitivity of the wild type, two atairp2 mutant alleles (atairp2-1 and atairp2-2), and AtAIRP2 overexpressors (transgenic lines 10 and 19) during the germination stage. Germination rates were determined in the presence of different concentrations of NaCl (0, 75, 100, and 125 mm) as described above. Data represent means ± sd (n > 36) from three independent experiments. [See online article for color version of this figure.]

Figure 5.

Root growth and stomatal aperture of wild-type, atairp2, and 35S:AtAIRP2-sGFP plants in response to ABA treatment. A, Root-growth phenotypes of the wild type (WT), two atairp2 mutant alleles (atairp2-1 and atairp2-2), and AtAIRP2 overexpressors (transgenic lines 10 and 19) in response to different concentrations (0, 0.2, 0.4, and 0.8 μm) of ABA. Sterilized seeds were imbibed in water for 2 d and grown vertically on MS medium supplemented with the indicated concentrations of ABA for 10 d. Root growth patterns were monitored and analyzed using Scion Image software. Data represent means ± sd (n = 20). Bars = 0.5 cm. B, Stomatal aperture of the wild type, two atairp2 mutant alleles (atairp2-1 and atairp2-2), and AtAIRP2 overexpressors (transgenic lines 10 and 19) in response to different concentrations (0, 0.1, 1.0, and 10 μm) of ABA. Mature leaves from wild-type, atairp2 allele, and AtAIRP2-overexpressing plants were treated with a stomatal opening solution for 2 h and incubated with the indicated concentrations of ABA for 2 h. Stomata on abaxial surfaces were photographed by light microscopy. Bars = 10 μm. Stomatal aperture (the ratio of width to length) was quantified using at least 30 guard cells from each sample. Data represent means ± sd (n = 30). [See online article for color version of this figure.]

Figure 6.

AtAIRP2 expression levels were closely associated with drought tolerance. A, atairp2 loss-of-function mutants were more sensitive to drought than were wild-type (WT) plants. Light-grown, 2-week-old wild-type and atairp2 mutant allele (atairp2-1 and atairp2-2) plants were further grown for 12 d under normal conditions but without irrigation. The water-stressed plants were irrigated, and their survival ratios were determined after 3 d of irrigation. B, Overexpression of AtAIRP2 conferred tolerance to drought stress. Light-grown, 2-week-old wild-type and 35S:AtAIRP2-sGFP (lines 10 and 19) plants were grown for 15 d without irrigation. Survival percentages were determined 3 d after irrigation. C, Water loss rates of detached rosette leaves. Mature rosette leaves from 2-week-old wild-type, atairp2 allele, and 35S:AtAIRP2-sGFP lines were detached, and their fresh weights were measured at the indicated time points. Water loss rates were calculated as the percentage of fresh weight of the excised leaves. Data represent means ± sd (n = 7) from eight independent experiments. D, H₂O₂ production in response to drought stress. Control and water-stressed rosette leaves from wild-type, atairp2-1, atairp2-2, and 35S:AtAIRP2-sGFP plants were stained with 100 μg mL⁻¹ DAB overnight. Levels of drought-induced H₂O₂ production were visualized as a dark brown color. [See online article for color version of this figure.]

Figure 7.

The positive role of AtAIRP2 in ABA induction of drought stress-related gene expression required SnRK protein kinase activity. A, ABA induction profiles of AtAIRP2 in wild-type (WT), abi1-1, snrk2.2, snrk2.3, and snrk2.6 single knockout mutant, and snrk2.2 snrk2.3 snrk2.6 triple mutant plants. Light-grown, 10-d-old wild-type and various snkr2 mutant seedlings were treated with 100 μm ABA. Total RNA was extracted from the treated tissues and analyzed by real-time qRT-PCR. RAB18 was a positive control for ABA induction, and UBC10 was used as a loading control. B, ABA induction profiles of drought-related genes in wild-type, atairp2-2, and AtAIRP2-overexpressing plants. Light-grown, 3-week-old plants were incubated with 100 μm ABA for 6 h. Induction patterns of various ABA- and drought-responsive genes (ABI1, ABI2, ABF3, ABF4, RD26, RD20, KIN2, and RAB18) were analyzed by real-time qRT-PCR. Data represent the fold induction of each gene by ABA (100 μm) relative to the control treatment (0 μm ABA). Mean values from three independent technical replicates were normalized to the levels of an internal control, glyceraldehyde-3-phosphate dehydrogenase C subunit mRNA. [See online article for color version of this figure.]

Figure 8.

Construction and characterization of atairp1/35S:AtAIRP2-sGFP and atairp2-2/35S:FLAG-AtAIRP1 complementation transgenic plants. A and B, RT-PCR and immunoblot analyses. AtAIRP2-sGFP and FLAG-AtAIRP1 fusion genes were ectopically expressed in atairp1 and atairp2-2 mutant plants, respectively. Transcript (A) and protein (B) levels of AtAIRP2-sGFP and FLAG-AtAIRP1 were examined in atairp1/35S:AtAIRP2-sGFP (lines 5 and 7) and atairp2-2/35S:FLAG-AtAIRP1 (lines 3 and 24) complementation T3 transgenic plants. Rubisco large subunit (RbcL) was used as a loading control. C, Phenotypic properties of atairp1/35S:AtAIRP2-sGFP and atairp2-2/35S:FLAG-AtAIRP1 complementation T3 transgenic plants during the germination stage. After imbibition in water for 2 d at 4°C, wild-type (WT), atairp1 and atairp2-2 mutant, and atairp1/35S:AtAIRP2-sGFP and atairp2-2/35S:FLAG-AtAIRP1 complementation T3 seeds were treated with different concentrations of ABA (0, 0.2, 0.4, and 0.8 μm) at 22°C under a 16-h-light/8-h-dark photoperiod. Germination percentages were determined in terms of cotyledon greening 7 d after germination. sd values were determined from four biological replicates (n = 40). Bars = 0.5 cm. D, Water stress tolerance of atairp1/35S:AtAIRP2-sGFP and atairp2-2/35S:FLAG-AtAIRP1 complementation T3 transgenic plants. Light-grown, 2-week-old wild-type, atairp1 and atairp2-2 mutant, and atairp1/35S:AtAIRP2-sGFP (lines 5 and 7) and atairp2-2/35S:FLAG-AtAIRP1 (lines 3 and 24) complementation T3 transgenic plants were further grown for 13 d without irrigation. Water-stressed plants were irrigated, and their survival ratios were determined after 3 d of irrigation. E, Water loss rates of detached rosette leaves. Mature rosette leaves from 2-week-old wild-type, atairp1 and atairp2-2 mutant, and atairp1/35S:AtAIRP2-sGFP (lines 5 and 7) and atairp2-2/35S:FLAG-AtAIRP1 (lines 3 and 24) complementation T3 transgenic plants were detached, and their fresh weights were measured at the indicated time points. Water loss rates were calculated as the percentage of fresh weight of the excised leaves. Data represent means ± sd (n = 7) from three independent experiments. [See online article for color version of this figure.]