The Arabidopsis RING finger E3 ligase RHA2b acts additively with RHA2a in regulating abscisic acid signaling and drought response

Abstract

We have previously shown that the Arabidopsis (Arabidopsis thaliana) RING-H2 E3 ligase RHA2a positively regulates abscisic acid (ABA) signaling during seed germination and postgerminative growth. Here, we report that RHA2b, the closest homolog of RHA2a, is also an active E3 ligase and plays an important role in ABA signaling. We show that RHA2b expression is induced by ABA and that overexpression of RHA2b leads to ABA-associated phenotypes such as ABA hypersensitivity in seed germination and seedling growth, enhanced stomatal closure, reduced water loss, and, therefore, increased drought tolerance. On the contrary, the rha2b-1 mutant shows ABA-insensitive phenotypes and reduced drought tolerance. We provide evidence showing that a rha2a rha2b-1 double mutant generally enhances ABA insensitivity of rha2b-1 in seed germination, seedling growth, and stomatal closure, suggesting that RHA2b and RHA2a act redundantly in regulating ABA responses. Genetic analyses support that, like RHA2a, the RHA2b action in ABA signaling is downstream of a protein phosphatase 2C, ABA-INSENSITIVE2 (ABI2), and in parallel with that of the ABI transcription factors ABI3/4/5. We speculate that RHA2b and RHA2a may have redundant yet distinguishable functions in the regulation of ABA responses.

Figure 1.

RHA2b exhibits E3 ligase activity. A, Alignment of deduced amino acid sequences of RHA2b and RHA2a. Conserved residues are highlighted in black, and Cys and His residues in the RING finger domain are underlined in black. The putative transmembrane domain is underlined in red, and basic amino acids in the RING finger domain are marked by cross stars. B, Subcellular localization of RHA2b and RHA2a. For 35S:GFP, 35S:AtAIB-GFP, 35S:RHA2a-GFP, or 35S:RHA2b-GFP, roots of 1-week-old seedlings were immersed in 2 μg mL⁻¹ DAPI solution for 10 to 15 min and then stained with 5 μg mL⁻¹ cold FM4-64 solution for 1 min. Before staining, the whole seedlings of 35S:RHA2a-GFP or 35S:RHA2b-GFP were pretreated with 50 μm MG132 for 6 h under dim-light conditions; for BRI1-GFP, roots of 1-week-old seedlings were stained with 5 μg mL⁻¹ cold FM4-64 solution for 1 min. All seedlings were observed with fluorescence microscopy. Bars = 30 μm. C, MBP-RHA2b fusion protein was assayed for E3 activity in the presence of E1 (from wheat [Triticum aestivum]), E2 (UBCh5b), and 6× His tag ubiquitin (Ub). The numbers at left denote the molecular masses of marker proteins in kD. MBP itself was used as a negative control. Samples were resolved by 8% SDS-PAGE. Nickel-horseradish peroxidase (HRP) was used to detect His tag ubiquitin (top panel), and the anti-MBP antibody was used for maltose fusion proteins (bottom panel).

Figure 2.

Comparison of RHA2b and RHA2a expression. A, pRHA2b:RHA2b-GUS and pRHA2a:RHA2a-GUS seedling staining. a and f, Two-day-old germinating seedlings. b and g, Seven-day-old seedlings. c and h, Fourteen-day-old seedlings. d and i, Primary roots of 7-d-old seedlings. e and j, Guard cells in leaves of 14-d-old seedlings. B, RHA2b and RHA2a expression pattern in dry seeds (DS) and during seed imbibition revealed by qRT-PCR. Imbibed seeds were kept in darkness at 4°C for 72 h and then transferred to medium with or without ABA (5 μm) in constant light at 22°C for germination. Total RNA was extracted at the indicated times (0 indicates the time immediately before transfer). Transcript levels were quantified by qRT-PCR against ACTIN2. Data shown are means ± sd of three independent biological determinations. C, ABA-induced RHA2b and RHA2a expression revealed by qRT-PCR. Two-week-old wild-type seedlings were treated with 50 μm ABA for 0, 0.5, 1, and 3 h. ACTIN2 primers were used as an internal control. Data shown are means ± sd of three independent biological determinations. Asterisks in B and C indicate significant differences from the corresponding control values determined by Student’s t test (* 0.01 ≤ P < 0.05, ** P < 0.01).

Figure 3.

ABA responses of Col-0, 35S:RHA2a, rha2a, 35S:RHA2b, rha2b-1, and rha2a rha2b-1 plants in seed germination and postgerminative growth. A, Seed germination percentage of the indicated genotypes grown on different concentrations of ABA was recorded at 3 d after the end of stratification. Data shown are means ± sd of three replicates. At least 100 seeds per genotype were measured in each replicate. B and C, Seed germination time course of the six genotypes grown on medium without ABA (B) or containing 0.5 μm ABA (C). Data shown are means ± sd of three replicates. At least 100 seeds per genotype were measured in each replicate. D, Photographs of young seedlings at 5 d after the end of stratification. Seeds were germinated and allowed to grow on horizontal agar medium containing 0, 0.2, and 0.5 μm ABA. E, Cotyledon greening percentage of the seedlings described in D. Values represent means ± sd of three replicates. At least 30 seedlings per genotype were measured in each replicate. F, Quantification of chlorophyll content. Seedlings described in D were collected for chlorophyll a/b extraction and measurement. Values represent means ± sd of three replicates. FW, Fresh weight of whole seedlings. Asterisks in E and F indicate significant differences from the corresponding wild-type values determined by Student’s t test (* 0.01 ≤ P < 0.05, ** P < 0.01). At least three independent experiments were conducted, and similar results were obtained.

Figure 4.

ABA responses of Col-0, 35S:RHA2a, rha2a, 35S:RHA2b, rha2b-1, and rha2a rha2b-1 plants in seedling growth. A, Sensitivity of seedlings to ABA. Four-day-old seedlings grown without ABA were transferred to a plate containing 20 μm ABA. The photographs were taken 11 d after the transfer. B, Measurement of taproot length. Taproot lengths of the seedlings in medium containing the indicated concentrations of ABA were measured (see “Materials and Methods”). Relative root growth compared with that on ABA-free medium is indicated. Data shown are means ± sd of three replicates. At least 30 seedlings per genotype were measured in each replicate. C, Quantification of lateral root number. D, Quantification of true leaf number. E, Measurement of seedling fresh weight. Seedlings were collected for measurement of fresh weight. Relative fresh weight compared with that on ABA-free medium is indicated. Asterisks in B to E indicate significant differences from the corresponding wild-type values determined by Student’s t test (* 0.01 ≤ P < 0.05, ** P < 0.01). At least three independent experiments were conducted, and similar results were obtained. [See online article for color version of this figure.]

Figure 5.

ABA responses of double mutant plants abi2-2 rha2a and abi2-2 rha2b-1 and triple mutant plants abi2-2 rha2a rha2b-1 in early seedling growth. A, Photographs of young seedlings grown horizontally on medium containing 0, 0.2, or 0.5 μm ABA for 5 d after the end of stratification. B, Cotyledon greening percentage of the seedlings described in A. Values represent means ± sd of three replicates. At least 30 seedlings per genotype were measured in each replicate. C, Quantification of chlorophyll content. Seedlings described in A were collected for chlorophyll a/b extraction and measurement. Values represent means ± sd of three replicates. FW, Fresh weight of whole seedlings. Asterisks in B and C indicate significant differences from the corresponding wild-type values determined by Student’s t test (* 0.01 ≤ P < 0.05, ** P < 0.01). At least three independent experiments were conducted, and similar results were obtained.

Figure 6.

Double mutant analysis between rha2b-1 and abi3-8, abi4-1, or abi5-7. Cotyledon greening percentage of the indicated genotypes grown on medium containing different concentrations of ABA was recorded at 5 d after the end of stratification. Data shown are means ± sd of three replicates. At least 30 seedlings per genotype were measured in each replicate. [See online article for color version of this figure.]

Figure 7.

Drought responses of Col-0, 35S:RHA2a, rha2a, 35S:RHA2b, rha2b-1, and rha2a rha2b-1 plants. A, Drought tolerance assay. Seven-day-old seedlings were transferred to soil for another 2 weeks (top row), subjected to progressive drought by withholding water for specified times (middle row), and then rewatered for 2 d (bottom row). B, Survival rate of the plants from A after rewatering; sd (error bars) was calculated from results of three independent experiments (n > 30 for each experiment). Asterisks indicate significant differences from the corresponding wild-type values determined by Student’s t test (* 0.01 ≤ P < 0.05, ** P < 0.01). C, Water loss rate. Leaves of the same developmental stage were excised and weighed at various time points after detachment. Values are means ± sd of three individual plants per genotype. Experiments were repeated at least three times with similar results.

Figure 8.

ABA-induced stomatal aperture of Col-0, 35S:RHA2a, rha2a, 35S:RHA2b, rha2b-1, and rha2a rha2b-1. A, Comparison of stomatal aperture in response to ABA. Epidermal peels from different genotype plants were kept for 12 h in the dark, incubated under light in stomata-opening solution for 1.5 h, and then treated with 0 and 50 μm ABA for 3 h before being observed by scanning electron microscopy. B, Measurement of stomatal aperture in response to ABA. The stomata width of epidermal peels from A was measured. Relative stomatal aperture compared with that on ABA-free medium is indicated. Data shown are means ± sd of three independent experiments (n = 40–50). Asterisks indicate significant differences from the corresponding wild-type values determined by Student’s t test (* 0.01 ≤ P < 0.05, ** P < 0.01).

Figure 9.

Expression of ABA- and stress-responsive genes. Expression of ABA- and stress-responsive genes was assayed by qRT-PCR in seedlings of Col-0, 35S:RHA2a, rha2a, 35S:RHA2b, rha2b-1, and rha2a rha2b-1. Two-week-old seedlings were treated with 100 μm ABA for 0, 3, and 6 h. Data shown are means ± sd of three independent experiments. Asterisks indicate significant differences from the corresponding wild-type values determined by Student’s t test (* 0.01 ≤ P < 0.05, ** P < 0.01).