Arabidopsis PHOSPHOTYROSYL PHOSPHATASE ACTIVATOR is essential for PROTEIN PHOSPHATASE 2A holoenzyme assembly and plays important roles in hormone signaling, salt stress response, and plant development

Abstract

PROTEIN PHOSPHATASE 2A (PP2A) is a major group of serine/threonine protein phosphatases in eukaryotes. It is composed of three subunits: scaffolding subunit A, regulatory subunit B, and catalytic subunit C. Assembly of the PP2A holoenzyme in Arabidopsis (Arabidopsis thaliana) depends on Arabidopsis PHOSPHOTYROSYL PHOSPHATASE ACTIVATOR (AtPTPA). Reduced expression of AtPTPA leads to severe defects in plant development, altered responses to abscisic acid, ethylene, and sodium chloride, and decreased PP2A activity. In particular, AtPTPA deficiency leads to decreased methylation in PP2A-C subunits (PP2Ac). Complete loss of PP2Ac methylation in the suppressor of brassinosteroid insensitive1 mutant leads to 30% reduction of PP2A activity, suggesting that PP2A with a methylated C subunit is more active than PP2A with an unmethylated C subunit. Like AtPTPA, PP2A-A subunits are also required for PP2Ac methylation. The interaction between AtPTPA and PP2Ac is A subunit dependent. In addition, AtPTPA deficiency leads to reduced interactions of B subunits with C subunits, resulting in reduced functional PP2A holoenzyme formation. Thus, AtPTPA is a critical factor for committing the subunit A/subunit C dimer toward PP2A heterotrimer formation.

Figure 1.

Creation of the P_AtPTPA::GUS fusion construct and the expression pattern of AtPTPA in Arabidopsis. A, AtPTPA promoter information. The 5′ sequence of AtPTPA from −1,721 to +21 bp was translationally fused to the GUS gene. B, Histochemical staining pattern of P_AtPTPA::GUS transgenic plants: a, rosette leaf; b, flowers and siliques; c, 6-d-old seedling; d, flower; e, stamen; f, root system; and g, stem. Red arrows indicate the newly emerged lateral roots, while the black arrow indicates the main root tip. C, RNA-blot analysis of AtPTPA in Arabidopsis tissues. Se, Seedlings; F, flowers; St, stem; L, rosette leaves; V, veins from leaves; R, roots. ACTIN2 was used as the loading control.

Figure 2.

Creation and analysis of amiR plants. A, The amiRNA sequence (top strand) was used to regulate the target sequence of AtPTPA (bottom strand). Solid lines indicate perfect matches, and dashed lines indicate mismatches. B, Leaf comparison between a wild-type plant (WT) and an amiR plant. Both plants were 4 weeks old. C, RNA-blot analysis of wild-type and amiR plants. A cDNA fragment of AtPTPA was used as the probe, and ACTIN2 was used as the RNA loading control. D, Western-blot analysis of wild-type and amiR plants. AtPTPA antibody was used to detect the AtPTPA protein, and cytosolic glyceraldehyde-3-phosphate dehydrogenase (GapC) was used as the protein loading control.

Figure 3.

The amiR plants are more sensitive to ABA than wild-type plants at germination and postgermination stages. A, Germination rates of wild-type (WT) and two independent amiR plants on MS plates supplemented with 1 µm ABA. B, Germination rates of wild-type, rcn1-6 mutant, and two independent amiR plants in the wild-type background on MS plates supplemented with different concentrations of ABA (1, 2, and 5 µm) on day 7. C, Relative main root length comparison (%) between ABA-treated and nontreated plants in wild-type and two independent amiR plants. Main root lengths were measured after 4-d-old seedlings were transferred onto MS plates supplemented with ABA (10 µm) or without ABA for 4 d. Error bars represent sd (n = 3). Asterisks indicate P < 0.01, as determined by Student’s t test.

Figure 4.

The amiR plants are more sensitive to salt, ethylene, and cantharidin treatments than wild-type plants. A, Relative root lengths of wild-type (WT), sos1-1 mutant, and two amiR plants in the wild-type background in the absence or presence of 100 mm NaCl. B, Relative hypocotyl lengths of wild-type, rcn1-6 mutant, amiR-4 (WT), amiR-6 (WT), and ein2-1 mutant plants in the absence or presence of 10 µm ACC. Plants were grown in darkness for 6 d before measurement. The mutant rcn1-6 was used as a negative control, and the mutant ein2-1 was used as a positive control. C, Relative root lengths of wild-type, rcn1-6 mutant, amiR-4 (WT), and amiR-6 (WT) plants in the absence or presence of the PP2A inhibitor cantharidin (Can; 10 µm). The mutant rcn1-6 was used as a negative control. Error bars represent sd (n = 3). Asterisks indicate P < 0.01, as determined by Student’s t test.

Figure 5.

Reduced expression of AtPTPA in the rcn1-6 background by the amiRNA technique leads to more sensitive phenotypes to ABA, NaCl, cantharidin, and ACC treatments. A, Phenotypes of wild-type (WT), rcn1-6 mutant, and three independent amiR plants in the rcn1-6 background, amiR-1 (rcn1-6), amiR-2 (rcn1-6), and amiR-3 (rcn1-6), in the absence (MS) or presence of ABA, NaCl, and cantharidin (Can), respectively. Measurements were taken 7 d after plants were moved to MS plates supplemented without or with ABA (1 µm), NaCl (100 mm), or cantharidin (10 µm). B, Phenotypes of wild-type, rcn1-6 mutant, amiR-1 (rcn1-6), and amiR-2 (rcn1-6) plants in the absence or presence of NaCl (100 mm). Photographs were taken 3 d after 4-d-old seedlings were transferred to MS plates without or with NaCl. C, Relative root lengths of wild-type, rcn1-6 mutant, amiR-1 (rcn1-6), and amiR-2 (rcn1-6) plants shown in B in the absence or presence of NaCl. D, Relative hypocotyl lengths of wild-type, rcn1-6 mutant, amiR-1 (rcn1-6), and amiR-2 (rcn1-6) plants in the absence or presence of ACC. The measurements were taken 6 d after stratified seeds were moved to room temperature in darkness with or without 10 µm ACC. Error bars represent sd (n = 3). Asterisks indicate P < 0.01, as determined by Student’s t test.

Figure 6.

Analysis of PP2A activity in wild-type, PP2A mutants, and amiR plants. WT, The wild type; amiR-6 (WT) and amiR-4 (WT), two independent amiR plants in the wild-type background; rcn1-6, pp2a-a1 mutant; rcn1-6 can10, pp2a-a1 mutant grown on MS plates containing cantharidin (10 µm); a1a2, pp2a-a1/pp2a-a2 double mutant; amiR-1 (rcn1-6) and amiR-2 (rcn1-6), two independent amiR plants in the rcn1-6 mutant background. PP2A activity, defined as pmol phosphate released per min per μg total proteins, was measured with plant materials grown on MS medium for 6 d. Error bars represent sd (n = 3). Asterisks indicate P < 0.01, as determined by Student’s t test.

Figure 7.

Characterization of the sbi1-2 mutant and immunoblot analysis for total and methylated PP2A-C subunits. A, Confirmation of the sbi1-2 mutant. Genotyping PCR was used to verify the homozygosity of the sbi1-2 mutant. LB, LP, and RP are three primers used in PCR experiments. The LP+RP combination was used to amplify the endogenous genomic SBI1 DNA fragment, whereas the LB+RP combination was used to amplify a partial T-DNA sequence and a partial SBI1 sequence in the T-DNA insertion mutant sbi1-2. B, Reverse transcription-PCR experiment to verify that sbi1-2 is a null mutant. ACTIN2 (ACT2) was used as the internal control. C, Analysis of PP2A activity in wild-type (WT), sbi1-2 mutant, rcn1-6 mutant, and amiR-4 (WT) plants. Plants were grown on MS medium for 6 d before samples were harvested for PP2A enzyme activity assay. Error bars represent sd (n = 3). Asterisks indicate P < 0.01, as determined by Student’s t test. D, Immunoblot analyses of steady-state levels of AtPTPA, total PP2Ac, methylated PP2Ac, and PP2A-A subunits in sbi1-2 mutant, wild-type, and amiR plants. Cytosolic glyceraldehyde-3-phosphate dehydrogenase (GapC) was used as the protein loading control. The relative abundance (compared with the wild type) for total PP2Ac , methylated PP2Ac, and cytosolic glyceraldehyde-3-phosphate dehydrogenase is shown below each band.

Figure 8.

Phenotypes of wild-type, rcn1-6 mutant, sbi1-2 mutant, and amiR plants after treatments with ABA, NaCl, cantharidin, and ACC. A, Relative root lengths (%) of wild-type (WT), rcn1-6 mutant, sbi1-2 mutant, and two amiR plants after treatments with ABA (10 µm) or NaCl (100 mm). Four-day-old seedlings were transferred to MS plates containing no ABA or NaCl or containing ABA or NaCl for 3 d before root length was measured. B, Relative root lengths on MS plates containing no cantharidin or MS plates containing cantharidin (Can; 2 and 10 µm) for 3 d before root length was measured. C, Relative hypocotyl lengths on MS plates containing no ACC or MS plates containing ACC (10 µm) for 3 d before root length was measured. The ethylene-insensitive mutant ein2-1 was used as a positive control for this assay. Error bars represent sd (n = 3). Asterisks indicate P < 0.01, as determined by Student’s t test.

Figure 9.

AtPTPA interacts with PP2A-A and PP2A-C subunits. A, Immunoprecipitation experiments to demonstrate that PP2A-A subunits interact with AtPTPA and PP2A-C subunits. PP2A-A antibodies were used as the pulling antibodies, and AtPTPA antibodies and PP2Ac antibodies were used in the western-blot experiments. Plant cellular extracts were from the wild type (WT), the rcn1-6 mutant, RCN1-YFP-overexpressing plants in the rcn1-1 background, and GFP-AtPTPA-overexpressing plants. Extracts from GFP-AtPTPA-overexpressing plants, but no pulling antibodies, were used as the negative control. Extracts from GFP-AtPTPA-overexpressing plants were loaded directly for the positive control on the blot. B, Pull-down experiments to demonstrate that GST-AtPTPA interacts with PP2A-C and PP2A-A subunits. Glutathione-agarose beads were used as the pulling matrix, and PP2Ac and PP2A-A antibodies were used in the western-blot experiments. Cellular extracts from wild-type and amiR-4 (WT) plants were used in pull-down experiments. GST alone was used as a negative control for the pull-down experiments, and extracts from the wild type were loaded directly in the western-blot experiment. Coomassie Brilliant Blue staining of pull-down mixtures is shown below.

Figure 10.

Methylation of PP2A-C subunits is dependent on PP2A-A subunits, and the protein-protein interaction between AtPTPA and PP2A-C subunits is PP2A-A dependent. A, Western-blot analyses of PP2A-A subunits, PP2Ac, methylated PP2Ac, and AtPTPA in the wild type (WT), pp2a-a1 mutant (i.e. rcn1-6), pp2a-a2/pp2a-a3 double mutant (a2a3), and pp2a-a1/pp2a-a2 double mutant (a1a2). Cytosolic glyceraldehyde-3-phosphate dehydrogenase (GapC) was used as the protein loading control. The relative abundance (compared with the wild type) for total PP2Ac, methylated PP2Ac, and cytosolic glyceraldehyde-3-phosphate dehydrogenase is shown below each band. B, Immunoprecipitation analysis of protein-protein interaction between PP2A-A subunit and PP2Ac. PP2A-A antibodies were used as the pulling antibodies, and PP2Ac antibodies were used in the western-blot experiment. Plant cellular extracts were from sbi1-2, wild-type, amiR-6 (WT), and amiR-4 (WT) plants in the immunoprecipitation experiment and cellular extracts (20 µg of proteins) from wild-type plants were loaded directly in the western-blot experiment. C, Pull-down experiments to show that the interaction between AtPTPA and PP2A-C subunits is PP2A-A dependent. Glutathione-agarose beads were used as the pulling matrix, and PP2Ac and PP2A-A antibodies were used in the western-blot experiments. Cellular extracts from wild-type, pp2a-a1 mutant (rcn1-6), and pp2a-a1 pp2a-a1/PP2A-A2 pp2a-a2 mutant (a1^−/−a2^+/−) plants were used in pull-down experiments. GST alone was used as a negative control for the pull-down experiments, and extracts from the wild type were loaded directly in western-blot experiments. Coomassie Brilliant Blue staining of pull-down mixtures is shown below.

Figure 11.

Pull-down experiments to show differential binding of PP2A-B subunits to PP2Ac. A, The PP2A-B subunit ATB′ η preferentially binds to methylated PP2Ac. B, The PP2A-B subunit ATB′ γ binds to both methylated and unmethylated PP2Ac. Cellular extracts from sbi1-2, rcn1-6, wild-type (WT), and amiR-4 (WT) plants were used in the pull-down experiments, and PP2Ac antibody was used in the western-blot experiment. Cellular extracts from the wild type were loaded in the western-blot analysis. Coomassie Brilliant Blue staining of pull-down mixtures is shown below. The relative abundance (compared with the wild type) for total PP2Ac is shown below each band.

Figure 12.

Model of the function of AtPTPA in the assembly of the PP2A holoenzyme in plants. AtPTPA, together with an A subunit of PP2A, binds to a PP2A-C subunit and converts it into a form that can accept a B subunit or be methylated by SBI1 before accepting a B subunit. The PP2A holoenzyme containing a methylated C subunit is more active (higher activity) than the one with a C subunit without methylation (lower activity).