The Arabidopsis RING E3 ubiquitin ligase AtAIRP3/LOG2 participates in positive regulation of high-salt and drought stress responses

Abstract

Really Interesting New Gene (RING) E3 ubiquitin ligases have been implicated in cellular responses to the stress hormone abscisic acid (ABA) as well as to environmental stresses in higher plants. Here, an ABA-insensitive RING protein3 (atairp3) loss-of-function mutant line in Arabidopsis (Arabidopsis thaliana) was isolated due to its hyposensitivity to ABA during its germination stage as compared with wild-type plants. AtAIRP3 contains a single C3HC4-type RING motif, a putative myristoylation site, and a domain associated with RING2 (DAR2) domain. Unexpectedly, AtAIRP3 was identified as LOSS OF GDU2 (LOG2), which was recently shown to participate in an amino acid export system via interaction with GLUTAMINE DUMPER1. Thus, AtAIRP3 was renamed as AtAIRP3/LOG2. Transcript levels of AtAIRP3/LOG2 were up-regulated by drought, high salinity, and ABA, suggesting a role for this factor in abiotic stress responses. The atairp3/log2-2 knockout mutant and 35S:AtAIRP3-RNAi knockdown transgenic plants displayed impaired ABA-mediated seed germination and stomata closure. Cosuppression and complementation studies further supported a positive role for AtAIRP3/LOG2 in ABA responses. Suppression of AtAIRP3/LOG2 resulted in marked hypersensitive phenotypes toward high salinity and water deficit relative to wild-type plants. These results suggest that Arabidopsis RING E3 AtAIRP3/LOG2 is a positive regulator of the ABA-mediated drought and salt stress tolerance mechanism. Using yeast (Saccharomyces cerevisiae) two-hybrid, in vitro, and in vivo immunoprecipitation, cell-free protein degradation, and in vitro ubiquitination assays, RESPONSIVE TO DEHYDRATION21 was identified as a substrate protein of AtAIRP3/LOG2. Collectively, our data suggest that AtAIRP3/LOG2 plays dual functions in ABA-mediated drought stress responses and in an amino acid export pathway in Arabidopsis.

Figure 1.

Isolation of the atairp3/log2-2 T-DNA-inserted loss-of-function mutant line. A, Screening of an ABA-hyposensitive RING E3 Ub ligase mutant in the germination stage. Wild-type (WT) and knockout mutant seeds were germinated on MS growth medium in the absence (top panel) or presence (bottom panel) of 0.5 μm ABA. Germination rates with respect to cotyledon greening were determined after 5 d. The atairp1 and atairp2 mutants served as positive controls for ABA-insensitive phenotypes, while the #99 RING mutant served as a negative control to demonstrate the wild-type phenotype in response to ABA. Bars = 1 cm. B, Schematic structure of the atairp3/log2-2 mutant line (SAIL_729_A08). Gray bars represent the 5′ and 3′ untranslated regions, black bars show the coding regions, and solid lines indicate the introns of AtAIRP3/LOG2 (GenBank accession no. NC_003074). The atairp3/log2-2 mutant contains double T-DNA insertions in the first introns after nucleotides 873 and 887 in an antisense orientation. White boxes depict T-DNA insertions. Primers used for genotyping PCR and RT-PCR are shown with arrows. Nucleotide sequences of the primers are listed in Supplemental Table S1. C, Genotyping and RT-PCR analyses of the atairp3/log2-2 knockout mutant and 35S:AtAIRP3-RNAi knockdown transgenic plants. Left panel, genotyping PCR of wild-type and atairp3/log2-2 plants. Primers used for genomic PCR are shown on the right side of the agarose gel. Middle panel, RT-PCR of wild-type and atairp3/log2-2 plants. Primers used for RT-PCR are shown on the right side of the gel. The level of Arabidopsis AtUBC10 (E2 ubiquitin-conjugating enzyme) transcripts was used as a loading control. Right panel, RT-PCR of wild-type, atairp3/log2-2, and 35S:AtAIRP3-RNAi (independent transgenic T4 lines 1 and 2) plants. Primers used for RT-PCR are shown on the right. AtUBC10 was used as a loading control. D, Schematic structure of the AtAIRP3/LOG2 gene and its predicted protein. Gray bars represent 5′ and 3′ untranslated regions, black bars show coding regions, and solid lines indicate introns. A putative N-terminal myristoylation site, a DAR2 domain, and a single C3HC4-type RING motif are indicated. Primers used for RT-PCR are shown with arrows. [See online article for color version of this figure.]

Figure 2.

Induction of AtAIRP3/LOG2 in response to drought, high salinity, and ABA. A, RT-PCR analysis of AtAIRP3/LOG2. Light-grown, 10-d-old Arabidopsis seedlings were subjected to drought (0, 1.5, 3, and 6 h), 300 mm NaCl (0, 1.5, and 3 h), or 100 μm ABA (1.5 and 3 h). Total RNA prepared from the treated tissues was analyzed by RT-PCR using gene-specific primer sets. RD29A served as a positive control for drought and salt treatments, whereas RAB18 served as a positive control for ABA induction. AtUBC10 was used as a loading control. B, Real-time qRT-PCR analysis of AtAIRP3/LOG2. Total RNA was isolated from the treated tissues and used for real-time qRT-PCR. Fold induction of AtAIRP3/LOG2 was normalized to levels of glyceraldehyde-3-phosphate dehydrogenase C subunit mRNA, which served as an internal control. Bars indicate means ± sd from three independent experiments. C, AtAIRP3/LOG2 promoter-GUS assay. Light-grown, 10-d-old AtAIRP3-promoter:GUS transgenic T3 plants were grown under drought conditions (4 h) with 300 mm NaCl (4 h) or with 100 μm ABA (4 h). Histochemical localization of GUS activities in leaf and root tissues was visualized by 5-bromo-4-chloro-3-indolyl β-D-glucoside staining for 1 h. Bars = 50 μm. [See online article for color version of this figure.]

Figure 3.

ABA-insensitive phenotypes of atairp3/log2-2 knockout mutant and 35S:AtAIRP3/LOG2-RNAi knockdown transgenic plants with respect to germination rates and stomatal movements. A, ABA-mediated inhibition of germination. Wild-type (WT), atairp3/log2-2 mutant, and 35S:AtAIRP3-RNAi (T4 lines 1 and 2) seeds were germinated on MS growth medium supplemented with different concentrations (0, 0.2, 0.5, and 1.0 μm) of ABA. Germination rates were determined after 3 to 5 d. Photographs were taken at 5 d after germination. Bars = 1 cm. B, Germination percentages of wild-type, atairp3/log2-2 mutant, and 35S:AtAIRP3-RNAi (T4 lines 1 and 2) seeds in response to ABA. Top panel, germination percentages were determined with respect to radicle emergence at 3 d after ABA treatment. Bottom panel, germination percentages were measured in terms of cotyledon greening at 5 d after ABA treatment. Bars indicate means ± sd (n = 110) from three independent experiments. C, ABA-mediated stomatal closure. Light-grown, 4-week-old rosette leaves of wild-type, atairp3/log2-2 mutant, and 35S:AtAIRP3-RNAi (T4 lines 1 and 2) plants were immersed in stomatal opening solution for 4 h and in ABA solution (0, 0.1, 1.0, or 10 µm) for another 2 h. Bright-field microscopy was used to photograph the guard cells. Bars = 10 μm. D, Measurement of stomatal aperture after ABA treatment. Stomatal apertures of wild-type, atairp3/log2-2 mutant, and 35S:AtAIRP3-RNAi (T4 lines 1 and 2) leaves were measured as the ratio of width to length after ABA treatment. Bars indicate means ± sd (n ≥ 35). [See online article for color version of this figure.]

Figure 4.

Cosuppression and complementation experiments. A and B, Arabidopsis plants that expressed AtAIRP3/LOG2 under the control of the CaMV 35S promoter were constructed. Several independent T4 transgenic plants (35S:AtAIRP3/LOG2) were obtained and used for phenotypic assays. A, Inhibition of germination by ABA in wild-type (WT), atairp3/log2-2 mutant, and 35S:AtAIRP3/LOG2 transgenic T4 (independent lines 1 and 2) plants. Freshly harvested seeds of each plant were germinated on MS growth medium supplemented with 0.5 μm ABA. Percentages of green cotyledon development were counted after 5 d. B, AtAIRP3/LOG2 transcript levels of wild-type and 35S:AtAIRP3/LOG2 transgenic plants. AtUBC10 was used as an equal loading control. C to E, Generation and phenotypic analysis of atairp3/AtAIRP3 complementation transgenic plants. C, Cartoon of the AtAIRP3/LOG2 complementation construct. The plasmid construct (3.2 kb in length) is composed of a 1.35-kb upstream region and a 1.85-kb coding region. The gray bar indicates the upstream region. The black bars indicate coding regions, and the white bar indicates the 5′ untranslated region. Solid lines represent introns. D, Transcript levels of wild-type, atairp3/log2-2, and atairp3/AtAIRP3 T3 complementation transgenic plants. Total RNAs were isolated as indicated and subjected to RT-PCR analysis using gene-specific primer sets (Supplemental Table S1). E, Complementation of atairp3/log2-2 by the AtAIRP3/LOG2 transgene. Seeds of wild-type, atairp3/log2-2, and atairp3/AtAIRP3 complementation T3 (independent lines 1–4) plants were germinated on MS medium containing 0.5 μm ABA. Percentages of green cotyledon development of wild-type, atairp3/log2-2, and atairp3/AtAIRP3 complementation T3 plants were determined after 5 d. Bars are means ± sd (n = 72) from three independent experiments. [See online article for color version of this figure.]

Figure 5.

Hypersensitive phenotypes of atairp3/log2-2 knockout mutant and 35S:AtAIRP3/LOG2-RNAi knockdown transgenic plants in response to high salinity. A, NaCl-sensitive phenotypes of atairp3/log2-2 and 35S:AtAIRP3/LOG2-RNAi plants as compared with wild-type (WT) plants. Wild-type, mutant, and RNAi (independent T4 lines 1 and 2) seedlings were grown for 5 d under normal growth conditions and placed in medium supplemented without (top panel) or with (bottom panel) 150 mm NaCl. The morphological abnormalities were observed after 14 d. Some of the atairp3/log2-2 mutant leaves were severely discolored and became whitish or dark brown, as indicated by red and brown arrows, respectively. Bars = 1 cm. B, Chlorophyll content of leaves from wild-type, mutant, and RNAi transgenic plants after salt treatment. Wild-type, atairp3/log2-2, and 35S:AtAIRP3/LOG2-RNAi plants were identically treated as described above, and total leaf chlorophyll levels were measured. Bars indicate means ± sd from three independent experiments. C, Root growth analysis of wild-type, atairp3/log2-2, and 35S:AtAIRP3-RNAi plants in response to different concentrations (0, 100, and 150 mm) of NaCl. Wild-type, mutant, and RNAi (independent T4 lines 1 and 2) seedlings were grown for 5 d under normal growth conditions and placed in medium supplemented without (left panel) or with (right panel) 100 to 150 mm NaCl. The root growth profiles were observed after 14 d. Note that lateral root formation in atairp3/log2-2 and RNAi plants was greatly reduced by high salinity relative to wild-type plants. D, Evans blue staining. Mature, healthy leaves from wild-type and atairp3/log2-2 mutant plants were incubated with 300 mm NaCl for 6 to 12 h. Salt-treated leaves were subsequently treated with Evans blue staining solution. The degree of cell death was determined by the strength of dark blue color. Bar = 1 cm. [See online article for color version of this figure.]

Figure 6.

Phenotypic analysis of atairp3/log2-2 knockout mutant and 35S:AtAIRP3/LOG2-RNAi knockdown transgenic plants in response to drought stress. A, Hypersensitive phenotypes of atairp3/log2-2 and 35S:AtAIRP3/LOG2-RNAi plants in response to water deficit. Wild-type (WT), mutant, and RNAi (independent T4 transgenic lines 1 and 2) plants were grown for 3 weeks in pots under normal growth conditions. The plants were grown for another 14 d without watering. Survival of these water-stressed plants was monitored 3 d after rewatering. B, Measurement of leaf water loss rates. The aerial parts of 4-week-old wild-type, atairp3/log2-2, and 35S:AtAIRP3/LOG2-RNAi plants were incubated at room temperature for different time periods. Changes in the fresh weight of leaves were measured at the given time points. Bars indicate means ± sd of 10 plants. Similar results were obtained from four biologically independent replicates. [See online article for color version of this figure.]

Figure 7.

AtAIRP3/LOG2 interacts with RD21. A, Yeast two-hybrid screening using a cDNA library prepared from 3-d-old etiolated seedlings and a full-length AtAIRP3/LOG3 cDNA. Arabidopsis cDNAs were cloned into the pACT2 vector (prey), and AtAIRP3/LOG2 was cloned into the pGBKT7 vector (bait). The cDNA-pACT2 and AtAIRP3/LOG2-pGBKT7 plasmids were cotransformed into yeast AH109 cells. Yeast cells were plated on SD/−Trp/−Leu/−His medium that contained 10 mm 3-AT and allowed to grow for 5 d at 30°C. The yeast cells containing RD21-pACT2 + AtAIRP3/LOG2-pGBKT7 grew efficiently in SD/−Trp/−Leu/−His medium in the presence of 3-AT. p53 + T-antigen were used as the positive control. Lambda + T-antigen and p53 + RD21 were used as the negative control. OD₆₀₀, Optical density at 600 nm. B, Schematic representation of full-length (RD21 full), intermediate (RD21 ΔN and RD21 ΔC), mature (mRD21), and deletion (cRD21) forms of RD21. P, Pro-rich domain; S, signal peptide. C, Yeast two-hybrid assay. Different forms of RD21 were cloned into pGADT7. The RD21-pGADT7 and AtAIRP3/LOG2-pGBKT7 constructs were cotransformed into yeast AH109 cells. Yeast cells were grown in SD/−Leu/−Trp/−His/−Ade growth medium at 30°C for 3 d. p53 + T-antigen were used as the positive control. Lambda + T-antigen were used as the negative control. D, In vitro IP assay. Bacterially expressed AtAIRP3/LOG2-flag and mRD21-myc fusion proteins were coincubated in the presence of an anti-flag affinity gel matrix. After extensive washing, the bound proteins were eluted with Gly (100 mm) buffer and subjected to immunoblot analysis using anti-flag and anti-myc antibodies. E, In vivo IP assay. The AtAIRP3/LOG2-flag and mRD21-myc fusion genes were transiently expressed in tobacco leaves. Leaf crude extracts (1 mg of protein) were 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. [See online article for color version of this figure.]

Figure 8.

AtAIRP3/LOG2-dependent degradation of RD21. A, Cell-free degradation assay for mRD21. The mRD21-myc protein was incubated for 0 to 3 h with crude extracts of salt-treated (300 mm NaCl) 10-d-old wild-type or atairp3/log2-2 mutant seedlings in the absence (lanes labeled 0 h, 1.5 h, and 3 h) or presence (lanes labeled 3 h-M) of 50 μm MG132. The time-dependent protein levels were examined by immunoblotting with anti-myc antibody. RGA1, which is known to be regulated by the Ub-26S proteasome pathway, was used as a positive control for proteasome-dependent degradation. The RGA1-flag protein was detected with an anti-flag antibody. Rubisco served as a loading control and was detected by Ponceau S staining. The protein levels were quantified using Multi Gauge version 3.1 software (Fuji Film). B, In vitro ubiquitination of mRD21 by AtAIRP3/LOG2. Recombinant AtAIRP3/LOG2-flag or the AtAIRP3/LOG2-flag^C319S derivative was coincubated with mRD21-myc in the presence or absence of Ub, ATP, E1 (Arabidopsis UBA1), and E2 (Arabidopsis UBC8) at 30°C for 1 h. The reaction mixture was analyzed by immunoblotting with an anti-myc antibody. Black circles indicate the shifted high-molecular-mass ubiquitinated protein band.