Arabidopsis RGLG2, functioning as a RING E3 ligase, interacts with AtERF53 and negatively regulates the plant drought stress response

Abstract

Transcriptional activities of plants play important roles in responses to environmental stresses. ETHYLENE RESPONSE FACTOR53 (AtERF53) is a drought-induced transcription factor that belongs to the AP2/ERF superfamily and has a highly conserved AP2 domain. It can regulate drought-responsive gene expression by binding to the GCC box and/or the dehydration-responsive element in the promoter of downstream genes. Overexpression of AtERF53 driven by the cauliflower mosaic virus 35S promoter resulted in an unstable drought-tolerant phenotype in T2 transgenic Arabidopsis (Arabidopsis thaliana) plants. Using a yeast two-hybrid screen, we identified a RING domain ubiquitin E3 ligase, RGLG2, which interacts with AtERF53 in the nucleus. The copine domain of RGLG2 exhibited the strongest interacting activity. We also demonstrated that RGLG2 could move from the plasma membrane to the nucleus under stress treatment. Using an in vitro ubiquitination assay, RGLG2 and its closest sequelog, RGLG1, were shown to have E3 ligase activity and mediated AtERF53 ubiquitination for proteasome degradation. The rglg1rglg2 double mutant but not the rglg2 or rglg1 single mutant exhibited a drought-tolerant phenotype when compared with wild-type plants. AtERF53-green fluorescent proteins expressed in the rglg1rglg2 double mutants were stable. The 35S:AtERF53-green fluorescent protein/rglg1rglg2 showed enhanced AtERF53-regulated gene expression and had greater tolerance to drought stress than the rglg1rglg2 double mutant. In conclusion, RGLG2 negatively regulates the drought stress response by mediating AtERF53 transcriptional activity in Arabidopsis.

Figure 1.

The expression profile of AtERF53. A, RNA gel-blot analyses of AtERF53 and COR15B expression by abiotic stresses. Two-week-old seedlings were dried on Whatman 3MM paper (Dry) and treated with 300 mm NaCl (NaCl). B, GUS staining of AtERF53 promoter:GUS transgenic plants from different growth stages. GUS activity is shown in transgenic Arabidopsis seedlings grown on MS medium. Panels 1 to 5, One-week-old seedlings; panels 6 to 10, 2-week-old plants; panels 11 to 15, flowers and siliques of 30-d-old plants. Panels 1, 6, and 11 are samples under normal control conditions, and the rest of the samples are with 1 h of drought treatment and 30 min of rehydration. Panels 4, 5, 9, and 10 are root systems. C, Subcellular localization of the AtERF53 protein. AtERF53 cDNA was fused to GFP, and the construct was expressed in transgenic Arabidopsis under the control of the cauliflower mosaic virus 35S promoter. GFP fluorescence was observed in nuclei of Arabidopsis protoplasts. Bar = 10 μm.

Figure 2.

AtERF53 could not be detected in 35S:AtERF53/Col-0 transgenic plants. A, RNA gel-blot analysis of three overexpressed lines. B, Western-blot analysis of T3 transgenic Arabidopsis plants overproducing AtERF53. No AtERF53 protein was detected. Lane C shows the control GFP protein. WT, Wild type. C, Degradation of the AtERF53-GFP protein in the root elongation zone and cotyledons was inhibited by the MG132 proteasome inhibitor. Line 1 of 35S:AtERF53-GFP/Col-0 was used for MG132 treatment. BF, Bright field. Bars = 50 μm. D, Western-blot analysis of transgenic Arabidopsis plants overproducing AtERF53-GFP under mock and MG132 treatments.

Figure 3.

Identification of RGLG interaction with AtERF53 via a yeast two-hybrid analysis. A, Diagram of the specific domains of RGLG2. B, The five yeast clones transformed with the bait, AtERF53, and different lengths of RGLG2 growing on the selective medium and exhibiting β-galactosidase activity. a.a., Amino acids; AD, the pGADT7 vector; BD (for the DNA-binding domain), the pGBKT7 vector; Lam/BD, human lamin C fused to the pGBKT7 vector as a negative control. Every plate has the same labeling as the first labeled plate. C, Localization of the RGLG2 protein interaction domain with AtERF53 using yeast two-hybrid assays. β-Galactosidase activity was measured for each transformant. Error bars indicate sd (n = 3). The enzyme activity of the empty vector was defined as 1.0. The diagram for each construct is indicated in the left panel. D, Yeast cells transformed with RGLG1-AD and AtERF53-BD growing on the selective medium and exhibiting β-galactosidase activity.

Figure 4.

RGLG2 translocalizes into the nucleus under stress conditions and interacts with AtERF53 using the BiFC system. A, Full-length RGLG2 was fused with GFP driven by the cauliflower mosaic virus 35S promoter. Some GFP fluorescence signals were observed in the nuclei and some at the plasma membranes. The experiment was repeated three times, and at least 60 protoplasts were observed. Bar = 10 μm. B, The RGLG2-GFP observed in 35S:RGLG2-GFP transgenic plants translocalized from the plasma membrane into the nucleus under salt stress. The images were taken after the transgenic plants were soaked in salt solution (200 mm) for 1 h. Bar = 10 μm. C, Interaction between AtERF53 and RGLG2 by BiFC. The top panels show constructs of cCFP-RGLG2 and nVenus-AtERF53. YFP and RFP fluorescence, 4′,6-diamino-phenylindole (DAPI; for nuclear staining), bright-field, and merged images are shown for each kind of transformation combination. Bar = 25 μm.

Figure 5.

RGLG2 functions as an E3 ubiquitin ligase and mediates AtERF53 protein ubiquitination. A, In the presence of the ubiquitin, E1, and E2 enzymes (UBCH5c), RGLG2-GST fusion proteins displayed ubiquitin E3 ligase activity. Protein bands with ubiquitin attached were detected by an anti-ubiquitin immunoblot (IB) analysis (10% SDS-PAGE). B, Detection of RGLG2-GST autoubiquitination. RGLG2-GST fusion proteins were detected with a GST antibody, and shifted bands indicate the attachment of one or two ubiquitin molecules (10% SDS-PAGE). C, RGLG2 mediates the ubiquitination of the AtERF53 protein. The full-length AtERF53 protein was fused with His and Trx tags (His-AtERF53-Trx) and used as a substrate for the assay. Anti-Trx was used in the immunoblot analysis to detect the Trx-tagged substrate protein (10% SDS-PAGE).

Figure 6.

Phenotypic and stress tolerance studies of rglg1rglg2 double mutants. A, Phenotypes of 3-week-old wild-type plants (WT) and rglg1rglg2 double mutants. B, Survival rates of wild-type, rglg2 single mutant, and rglg1rglg2 double mutant plants after withholding water for 14 to 16 d (Drought) and rehydration for 4 d (Rehydration). Twenty plants (five plants per pot) were tested in each experiment. The survival rate is indicated below the rehydration panels. This experiment was repeated three times with similar results.

Figure 7.

Gene expression and protein detection in 35S:AtERF53-GFP/rglg1rglg2 transgenic plants. A, Relative transgene expression of AtERF53-GFP in 35S:AtERF53-GFP/Col-0, 35S:AtERF53-GFP/rglg1rglg2, and wild-type plants as measured by real-time PCR. Error bars indicate sd (n = 3). B, Protein gel-blot analysis of GFP or AtERF53-GFP protein levels in 35S:GFP/Col-0, 35S:AtERF53-GFP/Col-0, and 35S:AtERF53/rglg1rglg2 using the GFP antibody. The bottom arrow indicates the GFP protein band, and the top arrow indicates the AtERF53-GFP fusion protein band. C, Confocal microscopic observation of GFP fluorescence in roots of 35S:AtERF53/rglg1rglg2 (two independent lines) and 35S:AtERF53-GFP/Col-0 (two lines). Bars = 50 μm. D, Relative gene expression of COR15B and P5CS1 in 35S:AtERF53-GFP/Col-0, rglg1rglg2, 35S:AtERF53-GFP/rglg1rglg2, 35S:RGLG2/rglg1rglg2 (RGLG2OE), and wild-type (WT) plants as measured by real-time PCR. Error bars indicate sd (n = 3). E, Drought tolerance test of rglg1rglg2 double mutants (rglg1/2) and 35S:AtERF53-GFP/rglg1rglg2 (OE lines). Numbers under the panels indicate the survival over total plants. This experiment was repeated three times with similar results.

Figure 8.

A model of RGLG1 and RGLG2 regulating drought stress signaling by mediating AtERF53 degradation. During drought stress, AtERF53 is induced to activate downstream gene expression. It is possible that RGLG1 and RGLG2 are down-regulated or that the ubiquitination and proteolysis processes are blocked in the early hours of drought stress stimulus. Under normal conditions, AtERF53 is recognized and ubiquitinated by RGLG2 and RGLG1 via an unknown posttranslational modification and then subjected to 26S proteasome proteolysis. The AtERF53-mediated drought stress response is thereby prevented.