The PP6 phosphatase regulates ABI5 phosphorylation and abscisic acid signaling in Arabidopsis

Abstract

The basic Leucine zipper transcription factor ABSCISIC ACID INSENSITIVE5 (ABI5) is a key regulator of abscisic acid (ABA)-mediated seed germination and postgermination seedling growth. While a family of SUCROSE NONFERMENTING1-related protein kinase2s (SnRK2s) is responsible for ABA-induced phosphorylation and stabilization of ABI5, the phosphatase(s) responsible for dephosphorylating ABI5 is still unknown. Here, we demonstrate that mutations in FyPP1 (for Phytochrome-associated serine/threonine protein phosphatase1) and FyPP3, two homologous genes encoding the catalytic subunits of Ser/Thr PROTEIN PHOSPHATASE6 (PP6), cause an ABA hypersensitive phenotype in Arabidopsis thaliana, including ABA-mediated inhibition of seed germination and seedling growth. Conversely, overexpression of FyPP causes reduced sensitivity to ABA. The ABA hypersensitive phenotype of FyPP loss-of-function mutants is ABI5 dependent, and the amount of phosphorylated and total ABI5 proteins inversely correlates with the levels of FyPP proteins. Moreover, FyPP proteins physically interact with ABI5 in vitro and in vivo, and the strength of the interaction depends on the ABI5 phosphorylation status. In vitro phosphorylation assays show that FyPP proteins directly dephosphorylate ABI5. Furthermore, genetic and biochemical assays show that FyPP proteins act antagonistically with SnRK2 kinases to regulate ABI5 phosphorylation and ABA responses. Thus, Arabidopsis PP6 phosphatase regulates ABA signaling through dephosphorylation and destabilization of ABI5.

Figure 1.

Phenotypic Characterization of FyPP1 and FyPP3 Loss- and Gain-of-Function Mutants in ABA Responses. (A) Germination and growth of Col, f1, and f3 seeds and seeds from self-pollinated f1^−/+f3 plants incubated on GM plates with 0 (top panels) or 1 μM ABA (bottom panels) for 5 d. Seeds from self-pollinated f1^−/+f3 plants were more sensitive to ABA than Col seeds. (B) Germination of Col, f1, and f3 seeds and seeds from self-pollinated f1^−/+f3 plants incubated on GM plates with varying concentrations of ABA (0, 0.5, 1, 2.5, and 5 μM) for 5 d. Seeds from self-pollinated f1^−/+f3 plants were more sensitive to ABA than Col. Germination percentages were determined from three independent experiments, with more than 100 seeds per line for each experiment. Values are means ± sd. (C) Greening of Col, f1, and f3 seeds and seeds from self-pollinated f1^−/+f3 plants incubated on GM plates with 0 or 1 μM ABA for 5 d. Seeds from self-pollinated f1^−/+f3 plants were more sensitive to ABA than Col seeds. Greening was determined with an average of >100 seeds from three independent experiments. Values are means ± sd. (D) Germination of Col, f1, and F3Ri/f1 seeds incubated on GM plates with varying concentrations of ABA (0, 0.5, 1, 2.5, and 5 μM) for 5 d. All GM plates contained 0.1% ethanol. Ethanol-induced F3Ri/f1 seeds were more sensitive to ABA than Col or f1 seeds. Germination percentage was determined from three independent experiments, with more than 150 seeds per line for each experiment. Values are means ± sd. (E) Greening of Col, f1, and F3Ri/f1 seeds incubated on GM plates (plus 0.1% ethanol) with 0 or 1 μM ABA for 5 d. Ethanol-induced F3Ri/f1 seeds were more sensitive to ABA than Col seeds or f1 seeds. Greening was determined with an average of >150 seeds from three independent experiments. Values are means ± sd. (F) Germination of Col, F1DN, F3DN, F1OE, and F3OE seeds incubated on GM plates with varying concentrations of ABA (0, 0.5, 1, 2.5, and 5 μM) for 5 d. F1DN and F3DN seeds were more sensitive to ABA, while F1OE and F3OE seeds were less sensitive to ABA than Col. Germination percentages were determined from three independent experiments, with more than 100 seeds per line for each experiment. Values are means ± sd. (G) Greening of Col, F1OE, F3OE, F1DN, and F3DN seeds incubated on GM plates with 0 or 1 μM ABA for 5 d. F1DN and F3DN seeds were more sensitive to ABA, while F1OE and F3OE were less sensitive to ABA than Col seeds. Greening was determined with an average of >100 seeds from three independent experiments. Values are means ± sd. Asterisks indicate the levels of statistical significance as determined by Student’s t test: *P < 0.01 versus Col.

Figure 2.

Genetic Interaction between ABI5 and FyPPs. (A) Germination and growth of seeds of Col, abi5, and self-pollinated f1^−/+ f3 and f1^−/+ f3 abi5 plants incubated on GM plates with 0 or 0.5 µM ABA for 5 d in white light. Seeds from self-pollinated f1^−/+f3 plants showed increased sensitivity to ABA, while abi5-1 seeds were insensitive to ABA compared with Col controls. Seeds from self-pollinated f1^−/+ f3 abi5-1 plants were insensitive to ABA, similar to abi5-1 seeds. (B) Loss of FyPP1 and FyPP3 activity enhances the ABA-related phenotypes of ABI5OE seeds. Top panel shows seedlings grown on GM plates without ABA. The ungerminated seeds (25.2%) from self-pollinated f1^−/+ f3 ABI5OE plants were further phenotyped and genotyped and confirmed to be f1 f3 ABI5OE homozygotes. Bottom panel shows the seedlings grown on GM plates containing 0.5 µM ABA. The germination and greening percentages of each line are shown at the bottom of each panel, and the number of seeds used for the calculation is shown in parentheses. (C) Growth of Col, F3OE, ABI5OE, and F3OE ABI5OE seeds on GM plates with 0 or 0.5 µM ABA. Overexpression of FyPP3 rescued the ABA hypersensitivity phenotype of ABI5OE seeds. The germination and greening percentages of each line are shown at the bottom of each panel, and the number of seeds used for the calculation is shown in parentheses.

Figure 3.

Protein–Protein Interactions between FyPPs and ABI5. (A) Diagram of the FyPP1 protein constructs used in Y2H assays. F1-NT, FyPP1 N-terminal region (amino acids 1 to 49); F1-CD, FyPP1 catalytic domain (amino acids 50 to 277); F1-CT, FyPP1 C-terminal region (amino acids 50 to 303). (B) ABI5 protein interacted with the F1-NT domain of FyPP1 protein in yeast cells. AD, B42 activation domain; BD, LexA DNA binding domain; EV, empty vector control. (C) LCI assays showing that when fused with nLUC, ABI5 and group I ABI5-like proteins (AREB3 and EEL), but not group II protein (ABF2), interacted with both cLUC-FyPP1 and cLUC-FyPP3 in plant cells. Values are means ± sd, n = 3. EVc, cLUC empty vector; EVn, nLUC empty vector. (D) Co-IP of ABI5 and FyPP1 or FyPP3 in plant cells. α-HA affinity matrix was used for immunoprecipitation (IP); α-HA and α-Myc antibodies were used for immunoblotting (IB). Input, total protein before immunoprecipitation. (E) BiFC assays showing that AB15 and FyPP3 interacted in the nucleus. ABI5-YFP^N and FyPP3-YFP^C fusion proteins were expressed in onion epidermal cells through cobombardment. No YFP signal was observed in onion cells cobombarded with the YFP^N (YFP protein N-terminal) and YFP^C (YFP protein C-terminal) control plasmids. The nuclei were stained by DAPI (4',6-diamidino-2-phenylindole, blue). Bars = 50 μM.

Figure 4.

FyPP Directly Dephosphorylates ABI5. (A) In vitro kinase assay of GST-ABI5b (Ser119-Gln190). In the absence of ABA, there was no detectable phosphorylated GST-ABI5b when treated with the plant extracts derived from Col, f1 f3, F1DN, F3DN, F1OE, and F3OE seedlings. After treatment with ABA, there were increased amounts of phosphorylated GST-ABI5b when incubated with the plant extracts derived from f1 f3, F1DN, and F3DN seedlings, in contrast with the reduced abundance of phosphorylated GST-ABI5b when incubated with the plant extracts derived from F1OE and F3OE seedlings. (B) In vitro dephosphorylation of GST-ABI5b by GST-FyPP3. GST-FyPP3 reversed the ABA-dependent dephosphorylation of GST-ABI5b treated with the plant extracts derived from f1 f3 seedlings. Increasing amounts of GST-FyPP3 decreased the amount of phosphorylated GST-ABI5b, while the inactive phosphatase (GST-FyPP3D81N) had no effect on the phosphorylation status of GST-ABI5b. A mutant form of ABI5 (GST-ABI5bS145A) was used as a negative control in the experiment. The amounts of GST-FyPP3 and GST-FyPP3D^81N proteins used in the assay are indicated by the numbers (0, 0.1, and 1 μg). (C) Schematic representation of the domain structure of the dephosphorylation mimic mutant ABI5A4 and the phosphorylation mimic mutant ABI5D4 used for the Y2H assays. The labeled Ser (S) and Thr (T) residues were mutated to Ala (A) or Asp (D), respectively. (D) Y2H assays between FyPP1 N-terminal region (F1NT) and various ABI5 mutants shown in (A). The phosphorylation mimic mutant ABI5D4 showed enhanced interaction with F1NT. Values are means ± sd; n = 3. Asterisks indicate the levels of statistical significance as determined by Student’s t test: *P < 0.02 versus F1NT–ABI5 interaction. EV, empty vector control. [See online article for color version of this figure.]

Figure 5.

Role of FyPP1 and FyPP3 in the Regulation of ABI5 Protein Stability. (A) Immunoblot assays showing that more ABI5 protein accumulated in seeds of self-pollinated f1^−/+ f3, F1DN, and F3DN lines but less accumulated in F1OE and F3OE seeds compared with Col seeds treated with ABA (1 μM) for various durations (0, 1, 2, 4, and 6 d). A total of 100 μg protein was loaded for each lane. All immunoblot assays were performed side-by-side under identical conditions. Arrows indicate the ABI5 protein bands. RPT5 was used as a loading control. (B) Immunoblot assays showing that after ABA removal, ABI5 protein was more stable in F3Ri/f1, F1DN, and F3DN lines but more rapidly degraded in F1OE and F3OE seeds compared with Col seeds. The seeds were treated with ABA (5 μM) in white light for 3 d and then harvested at different time points after removal of the ABA. The medium used for each line is indicated on the top of each panel. abi5-1 mutant seeds were used as a negative control in the experiment. A total of 100 μg protein was loaded for each lane. All immunoblot assays were performed side-by-side under identical conditions. RPT5 was used as a loading control. (C) Quantitative RT-PCR assay showing that the expression of RD29A and RD29B was hyperinduced in f1 f3, F1DN, and F3DN seedlings treated with ABA compared with ABA-treated Col. Five-day-old seedlings were incubated in 1× Murashige and Skoog liquid medium with ABA (100 μM) or control solvent (DMSO) for 1.5 h before harvest. Values are means ± sd; n = 3. Asterisks indicate the levels of statistical significance as determined by Student’s t test: *P < 0.01 versus Col. [See online article for color version of this figure.]

Figure 6.

Antagonistic Relationship between OST1 (SnRK2.6) and FyPPs. (A) Germination and growth of Col, ost1, F3DN, and ost1 F3DN seeds and seeds from self-pollinated f1^−/+ f3 and f1^−/+ f3 ost1 plants incubated on GM plates with or without ABA. Seeds were incubated under white light for 5 d. (B) Germination of Col, ost1, F3DN, and ost1 F3DN seeds and seeds from self-pollinated f1^−/+ f3 and f1^−/+ f3 ost1 plants. Seeds were grown under white light for 5 d on GM plates with the indicated concentrations of ABA. Germination was determined with an average of >100 seeds from three independent experiments. Values are means ± sd. (C) Greening of Col, ost1, F3DN, and ost1 F3DN seeds and seeds from self-pollinated f1^−/+ f3 and f1^−/+ f3 ost1 plants incubated on GM plates with (1 μM) or without ABA treatment for 5 d. Greening was determined with an average of >100 seeds from three independent experiments. Values are means ± sd. Asterisks indicate the levels of statistical significance as determined by Student’s t test: *P < 0.01 versus Col. (D) Immunoblot analysis of ABI5 accumulation in kinase- and phosphatase-deficient mutants. In the absence of ABA, there was no detectable ABI5 protein in Col, ost1, f1^−/+ f3, F3DN, f1^−/+ f3 ost1, and F3DN ost1 seedlings. After ABA treatment (1 μM, 8 d), ABI5 (indicated by the arrows) hyperaccumulated in seeds of self-pollinated f1^−/+ f3 or F3DN lines, in contrast with the reduced accumulation of ABI5 in seeds of self-pollinated f1^−/+ f3 ost1 plants and F3DN ost1 lines. There was no detectable ABI5 protein in Col and ost1 seedlings at this stage. The arrowhead indicates the nonspecific band recognized by the ABI5 antibody. Arrows indicate the ABI5 bands. RPT5 was used as a loading control. A total of 100 μg protein was loaded for each lane.

Figure 7.

Degradation of FyPP1 and FyPP3 Protein upon ABA Treatment. (A) YFP-FyPP1 protein levels in seedlings treated with control solvent (DMSO) for different durations (0, 1, 2, 5, 8, and 24 h). (B) YFP-FyPP3 protein levels in seedlings treated with control solvent (DMSO) for different durations (0, 1, 2, 5, 8, and 24 h). (C) YFP-FyPP1 protein levels in seedlings treated with ABA for different durations (0, 1, 2, 5, 8, and 24 h). (D) YFP-FyPP3 protein levels in seedlings treated with ABA for different durations (0, 1, 2, 5, 8, and 24 h). The relative amounts of YFP-FyPP proteins are shown below the immunoblot assays; 100 μg total protein was loaded for each lane.

Figure 8.

A Model Showing the Antagonistic Interaction between SnRK2 Kinases and FyPP/PP6 Phosphatases in ABA Signaling. In the presence of ABA (+ABA), ABA mediates the formation of an ABA receptor-ABA-PP2C phosphatase complex (such as Pyr-ABA-PP2C complex). This causes the autophosphorylation and activation of the downstream SnRK2 kinases, including SnRK2.2, SnRK2.3, and SnRK2.6. Meanwhile, ABA promotes the degradation of the PP6 catalytic subunits, including FyPP1 and FyPP3. The active SnRK2 kinases then phosphorylate and activate the downstream transcription factors, such as ABI5, which further activate the expression of ABRE-containing genes and repress seed germination. By contrast, in the absence of ABA (-ABA), PP2C phosphatases persist, allowing them to inhibit SnRK2 kinase activity by forming PP2C-SnRK2 protein complexes and dephosphorylating SnRK2 kinases. Simultaneously, FyPP/PP6 perceives this signal and dephosphorylates ABI5, triggering its degradation and altering the expression of ABRE-containing genes, therefore promoting seed germination. The red lines indicate events happening in the presence of ABA. The green lines indicate events happening in the absence of ABA. The dashed lines indicate events happening through unknown mechanisms. p, phosphate.