SDIR1 is a RING finger E3 ligase that positively regulates stress-responsive abscisic acid signaling in Arabidopsis

Abstract

Ubiquitination plays important roles in plant hormone signal transduction. We show that the RING finger E3 ligase, Arabidopsis thaliana SALT- AND DROUGHT-INDUCED RING FINGER1 (SDIR1), is involved in abscisic acid (ABA)-related stress signal transduction. SDIR1 is expressed in all tissues of Arabidopsis and is upregulated by drought and salt stress, but not by ABA. Plants expressing the ProSDIR1-beta-glucuronidase (GUS) reporter construct confirmed strong induction of GUS expression in stomatal guard cells and leaf mesophyll cells under drought stress. The green fluorescent protein-SDIR1 fusion protein is colocalized with intracellular membranes. We demonstrate that SDIR1 is an E3 ubiquitin ligase and that the RING finger conservation region is required for its activity. Overexpression of SDIR1 leads to ABA hypersensitivity and ABA-associated phenotypes, such as salt hypersensitivity in germination, enhanced ABA-induced stomatal closing, and enhanced drought tolerance. The expression levels of a number of key ABA and stress marker genes are altered both in SDIR1 overexpression and sdir1-1 mutant plants. Cross-complementation experiments showed that the ABA-INSENSITIVE5 (ABI5), ABRE BINDING FACTOR3 (ABF3), and ABF4 genes can rescue the ABA-insensitive phenotype of the sdir1-1 mutant, whereas SDIR1 could not rescue the abi5-1 mutant. This suggests that SDIR1 acts upstream of those basic leucine zipper family genes. Our results indicate that SDIR1 is a positive regulator of ABA signaling.

Figure 1.

Expression Patterns of SDIR1. (A) Expression patterns of SDIR1 gene transcripts in response to drought, 300 mM NaCl, and 50 μM ABA treatment. Twelve micrograms of total RNA from each sample was hybridized with α-³²P-labeled SDIR1 probe. The 28s rRNA is shown as a loading control, and numbers below each lane indicate the relative expression ratio. (B) Expression of the SDIR1 gene in different tissues of Arabidopsis plants. Total RNA was isolated from various tissues (root, leaf, stem, flower, and silique) of 4-week-old wild-type plants grown under long-day growth conditions. RT-PCR was performed with either SDIR1-specific primers (top gel) or actin-specific primers (bottom gel). (C) SDIR1 promoter–GUS expression pattern in transgenic Arabidopsis plants. (a) One-day-old germinating seed. (b) Two-day-old germinating seedling. (c) Three-day-old seedling. (d) Four-day-old seedling. (e) Flowers. (f) Silique. (g) Guard cells. (h) Tissue localization of enhanced GUS expression in SDIR1 promoter–GUS transgenic seedlings treated with 300 mM NaCl for 5 h and drought for 5 h. (i) and (j) Details of GUS expression in guard cells under drought treatment. (i) Control without drought treatment. (j) Samples treated with drought for 3 h.

Figure 2.

Intracellular Membrane Localization of the SDIR1 Protein. (A) Amino acid sequence of SDIR1. Two putative transmembrane domains predicted by the protein domain analysis program SMART are shaded. The highly conserved RING finger domain is underlined, and asterisks indicate conserved Cys and His residues. Closed triangles indicate the transmembrane domain deletion positions. (B) Cell fractionation assays of SDIR1 and SDIR1 transmembrane domain deletion (SDIR1ΔTM) proteins. Total extract (T) of N. benthamiana leaf cells expressing a myc-vector control (CK) and myc-SDIR1 and myc-SDIR1ΔTM fusions was fractionated into soluble (S) and microsomal (M) fractions, and the myc fusion proteins were detected using an anti-myc antibody (top panel). The arrow indicates myc-SDIR1, and the triangle indicates myc-SDIR1ΔTM. Ponceau S staining of the transferred membrane is displayed as a loading control (bottom panel). (C) Further fractionation analysis for membrane association. T, total extract; S, soluble fraction; M, membrane fraction; B, buffer-extracted fraction; SD, SDS-extracted fraction; F, final membrane fraction. The antibody and loading controls are the same as in (B). (D) Subcellular localization of SDIR1 protein by GFP fusion expression in onion epidermal cells. Cells were analyzed by fluorescence microscopy and photographed after 16 h of incubation following bombardment. Panels from left to right: GFP control, GFP-SDIR1, GFP-SDIR1 treated with 1 M sucrose for plasmolysis, and GFP-SDIR1ΔTM (top panels). Samples were stained with 4′,6-diamidino-2-phenylindole; arrows indicate positions of nuclei (bottom panels).

Figure 3.

E3 Ubiquitin Ligase Activity of SDIR1 and the RING Mutant Variant. (A) Scheme of SDIR1 RING finger composition and the mutated amino acid in the RING finger. (B) E3 ubiquitin ligase activity of SDIR1. MBP-SDIR1 and its mutant form MBP-SDIR1 (H234Y) fusion proteins were assayed for E3 activity in the presence of E1 (from wheat), E2 (UBCh5b), and 6xHis tag ubiquitin (Ub). The numbers at left denote the molecular masses of marker proteins in kilodaltons. MBP itself was used as a negative control. Samples were resolved by 8% SDS-PAGE. The nickel–horseradish peroxidase was used to detect His tag ubiquitin (top panel), and the anti-MBP antibody was used for maltose fusion proteins (bottom panel).

Figure 4.

SDIR1 Structure, T-DNA Insertion Diagnostic PCR, and Phenotypes of SDIR1 Overexpression and Mutant Plants. (A) Schematic diagram of SDIR1 structure and T-DNA diagnostic PCR and RT-PCR. Closed boxes represent exons, and lines between closed boxes represent introns. P1, forward primer; P2, reverse primer; LBb1, primer specific to the T-DNA left border. RT Fw and RT Rev are primers used for RT-PCR analysis. aa, amino acids. (B) Diagnostic PCR of the T-DNA inserted in two different loci of SDIR1. DNA from homozygous insertion lines of sdir1-1 and sdir1-2 were used. M, molecular mass markers. Primers used for PCR are indicated above each lane. (C) RT-PCR analysis of the SDIR1 transcripts in wild-type and T-DNA insertion mutant seedlings. The primer pairs used for RT-PCR are shown in (A). ACTIN1 was used as an internal control. (D) Root phenotype of representative seedlings grown on vertical MS plates for 7 d (top panel). Bar = 1 cm. RNA blot and phenotype analysis of wild-type, two mutant, and 35S-SDIR1 plants. Ten micrograms of total RNA were loaded in each lane. 28S rRNA was used as an RNA-loading control (bottom panel). (E) Quantitative analysis of primary root length of wild-type, two mutant, and 35S-SDIR1 plants on MS medium at vertical growth position. The values are means ± sd (n = 30).

Figure 5.

Salt and Osmotic Sensitivity of 35S-SDIR1 and sdir1-1 Plants. (A) Quantitative analysis of germination on 100 mM NaCl. Germination (radical emergence) of wild-type, two mutant, and 35S-SDIR1 plants on MS medium containing 100 mM NaCl. Percentages are means (n = 60 to 90 each) of three repeats ± sd. (B) Growth phenotype of transgenic and mutant plants on MS medium containing 100 mM NaCl. Seeds were germinated and grown for 7 d. Numbers below the panel indicate the percentage of seedlings with green cotyledons in total seedlings (n = 60 to 90). (C) Osmotic effects on newly germinated seedling growth. Seeds of three different genotypes were germinated for 4 d on MS medium and MS medium containing 100 mM NaCl or 200 mM mannitol (Man), transferred to the same type of medium, and grown in a vertical position for an additional 1 d. Representative seedlings are shown.

Figure 6.

Responses to Drought of 35S-SDIR1, Wild-Type, and sdir1-1 Plants. (A) Drought tolerance assay of 3-week-old plants. Plants were grown in soil in the same container, withheld from water for 18 d (left), and then rewatered (right). The photographs were taken 1 d after rewatering. Representative plants from each treatment group are enlarged for better visualization. (B) Stomatal apertures of wild-type plants (left), 35S-SDIR1 transgenic plants (center), and sdir1-1 plants (right). Stomatal guard cells were observed in the middle of the watering period by liquid nitrogen–coupled scanning electron microscopy. (C) Measurement of stomatal aperture on transgenic and mutant seedlings corresponding to (B). Data are mean ratios of width to length ± se of three independent experiments (n = 30 to 40). (D) Transpiration rates. Leaves of the same developmental stages were excised and weighed at various time points after detachment. Each data point represents the mean of duplicate measurements. Error bars represent se (n = 8 each). (E) Effects of ABA on stomatal aperture in wild-type, 35S-SDIR1, and sdir1-1 plants. Epidermal peels from plants were kept for 12 h in the dark, incubated under light in buffer for 3 h, and then treated with 0 and 50 μM ABA for 4 h before aperture measurements. Data are mean ratios of width to length ± se of three independent experiments (n = 30 to 40).

Figure 7.

ABA Sensitivity of 35S-SDIR1 and sdir1-1 Plants. (A) Growth of different genotypes of plants on MS medium containing 1 μM ABA. Seeds were germinated and grown for 8 d. The percentages shown indicate seedlings with cotyledon expansion in total germinated seeds. (B) Growth of transgenic and mutant plants on MS medium containing a range of concentrations (0, 0.5, 1, 2, and 5 μM) of ABA. Seeds were germinated for 8 d on MS medium with or without ABA, and representative plants are shown. Bar = 1 cm. (C) ABA dose-response analysis of germination. Seeds were germinated for 4 d on plates containing different amounts of ABA. The germination percentage without ABA was considered to be 100%, and the germination frequency in ABA for these lines was normalized based on this value. Error bars represent se (triplicate measurements; n = 60). (D) ABA dose-response analysis of postgerminative growth (cotyledon greening/expansion). Results were scored at 10 d after plating (triplicate measurements; n = 60). The data analysis is the same as in (C).

Figure 8.

Expression of ABA and Stress-Responsive Genes in Wild-Type, 35S-SDIR1, and sdir1-1 Plants Induced by 100 μM ABA at Different Times. Total RNA was extracted from whole seedlings after different treatment times, as indicated. Numbers at top indicate the treatment period (hours). Total RNA (10 μg) was loaded in each lane and analyzed by RNA gel blots hybridized to gene-specific probes. The top panels show hybridization with the SDIR1 probe. The bottom panels show methylene blue–stained total RNA gels as a loading control. Group I genes were known as upstream genes in the ABA signaling pathway. Group II genes were ABA-responsive bZIP transcription factors. Group III showed the other stress- or ABA-responsive genes.

Figure 9.

Complementation Experiment. Numbers indicate different T3 homozygous transgenic lines. Photographs were taken after 10 d of germination. MS, control plate; ABA, MS plate supplied with 1 μM ABA. (A) Complementation of ABI5 in sdir1-1 mutant. (B) Complementation of SDIR1 in abi5-1 mutant. (C) Complementation of ABF3 in sdir1-1 mutant. (D) Complementation of ABF4 in sdir1-1 mutant.

Figure 10.

Proposed Model for the Role of SDIR1 in the ABA Signaling Pathway. Substrate modification could be either degradation or monoubiquitination.