Volume 7, Issue 6, Pages 960-976 (June 2014) Arabidopsis Protein Phosphatase 2C ABI1 Interacts with Type I ACC Synthases and Is Involved in the Regulation of Ozone-Induced Ethylene Biosynthesis  Ludwików Agnieszka , Cieśla Agata , Kasprowicz-Maluśki Anna , Mituła Filip , Tajdel Małgorzata , Gałgański Łukasz , Ziółkowski Piotr A. , Kubiak Piotr , Małecka Arleta , Piechalak Aneta , Szabat Marta , Górska Alicja , Dąbrowski Maciej , Ibragimow Izabela , Sadowski Jan   Molecular Plant  Volume 7, Issue 6, Pages 960-976 (June 2014) DOI: 10.1093/mp/ssu025 Copyright © 2014 The Authors. All rights reserved. Terms and Conditions

Figure 1 ABI1 Interacts with the C-Terminal Fragment of ACC Synthase 6 in Yeast. Interaction assay of ABI1 and ABI2 PP2Cs with full-length and the C-terminal fragment of ACS proteins. (A) Saturated cultures of the double-transformed yeast cultures were grown on double (DDO—SD medium without leucine and tryptophan), triple (TDO—SD medium without leucine, tryptophan, histidine), and quadruple (QDO—SD medium without leucine, tryptophan, histidine, adenine) selective medium. Representative examples are shown. (B) Schematic structures of ABI1, ABI2, and ACS proteins. The N-terminal deletion of ABI1 (∆N–ABI1) lacks amino acid residues 1–121. ∆–ACS2 and ∆–ACS6 lack the catalytic domain and comprise amino acid residues 464–496 and 471–495, respectively. Deleted protein fragments are indicated by open bars. Molecular Plant 2014 7, 960-976DOI: (10.1093/mp/ssu025) Copyright © 2014 The Authors. All rights reserved. Terms and Conditions

Figure 2 ABI1 Interacts with ACS6 and Dephosphorylates Its C-Terminal Fragment. (A) Analysis of the interaction between ABI1 and ABI2 with ACS2 and ACS6 by means of a BiFC assay in control (left) and MG132-treated (right) Arabidopsis thaliana protoplasts. Treatment with MG132 for 6h increases the BiFC signal for the ABI1–ACS2 interaction. The YFP fluorescent signal was located in the cytoplasm. Scale bar: 10 μm. (B) Pull-down assays to verify the interaction of ABI1 with ACS2 or ACS6 (upper panel), interaction of ABI2 with ACS2 or ACS6 (middle panel), and the GST/Strep fusion negative controls (lower panel). Input lines (upper/middle panels, lines 1 and 4; control panel, lines 2 and 3) represent 50% of the ABI1/2. Recombinant GST–ACS2, GST–ACS6 were pre-coupled to glutathione sepharose and incubated with StrepTag–ABI1 and StrepTag–ABI2, respectively. Pulled-down StrepTagged ABI1 or ABI2 proteins were detected (IB) with the epitope tag antibody. The presence of recombinant protein was confirmed using specific anti-ACS2 or anti-ACS6 antibodies. Note, for StrepTag–ABI1 isolated from T87 cell-suspension cultures, two sharp protein bands were observed that may arise from unknown protein modification. (C)32P-labeled GST–ACS6 was mixed with 3 μg of StrepTag–ABI1 purified from Arabidopsis T87 cell cultures subjected to various treatments. ACS phosphorylation level was visualized by autoradiography. ABA-inhibited StrepTag–ABI1 was used as a negative control. Western blot with anti-GST antibodies confirms the presence of the GST–ACS6 protein. The experiment was performed twice, with consistent results. (D) Densitometric analysis. The phospho-specific bands were normalized against the intensities of the corresponding 32P-labeled GST–ACS6 control band. Data are means ± SD of the relative band intensities from three independent experiments. Asterisks (**) indicate statistically significant changes at P < 0.001 from Student’s t-test. (E) Degradation of GST–ACS6 in wild-type and abi1td extracts. GST–ACS6 was incubated with 200 μg of protein extracts prepared from protoplasts incubated with or without 100 μM MG132 for 2h. GST–ACS6 protein level was examined by immunoblotting using anti-ACS6 antibody. Ponceau S staining confirms equal loading. Molecular Plant 2014 7, 960-976DOI: (10.1093/mp/ssu025) Copyright © 2014 The Authors. All rights reserved. Terms and Conditions

Figure 3 ABI1 Is a Negative Regulator of MPK6 Activity. (A) Phosphatase assays were performed in 100 μl final volume containing 5 μg recombinant GST–ABI1, 5 μg GST–PP2C6 or 2–3 μg of StrepTag–ABI1 purified from mock, (±)ABA (100 μM), and H2O2-treated (50mM) Arabidopsis cells. Each enzyme sample was assayed as three technical replicates. The results presented show average values of five independent biological replicates. (B) ABI1 inactivates MPK6 in vivo. Protoplasts from WT Arabidopsis Col-0 plants were transfected with 5 μg of plasmid DNA coding for MPK6–HA and StrepTag–ABI1 or StrepTag–ABI2. The MPK6 activity was analyzed using MBP as a substrate. Protein expression was confirmed by immunoblotting using anti-HA and anti-StrepTag antibodies. (C) ABI1 inactivates MPK6. For the analysis of kinase activity with MBP as a substrate, active MPK6 was combined with 3 μg of StrepTag–ABI1 protein purified from mock, ABA, or H2O2-treated Arabidopsis cell cultures, and recombinant GST–ABI1 or GST–PP2C6 as a negative control. Equal loading was confirmed by Coomassie staining of MBP. (D) ABI1 affects ACS phosphorylation by negative regulation of MPK6 activity. Active MPK6 was combined with 3 μg of StrepTag–ABI2 (mock) or StrepTag–ABI1 from mock, ABA-, or H2O2-treated Arabidopsis cells. MPK6 activity was analyzed directly with ACS2 and ACS6 as a substrate. (E, F) Reduction of MPK6 activity by ABI1 on MBP (E) and ACS6 as a substrate (F). The phospho-specific bands were quantified and then normalized by the intensities of the corresponding 32P-labeled MBP or 32P-labeled ACS6 control band, respectively. Equal loading was confirmed by Coomassie staining of MBP. Data are means ± SD of the relative band intensities from three independent experiments. An asterisk (*) indicates statistically significant changes from Student’s t-test. Molecular Plant 2014 7, 960-976DOI: (10.1093/mp/ssu025) Copyright © 2014 The Authors. All rights reserved. Terms and Conditions

Figure 4 Chemical Treatment Increases Ethylene Production, ACS Activities, and ACC Content in the abi1td Mutant. (A, B) The abi1td mutant showed increased ethylene production and total ACS activity in response to MG132 treatment. Total ACS enzyme activities were calculated after subtracting endogenous ACC in the crude protein extract. (C) The abi1td mutant shows increased ACC content in response to MG132. The amount of ACC was determined in total plant extracts as described in the ‘Methods’ section. (D) ACS6 activity is increased in the abi1td mutant. ACS6 activity was measured by the immune complex ACS assay using specific ACS6 antibodies. (E) Residual ACS2 activity in WT Col-0 and abi1td. ACS2 activity was measured by the immune complex ACS assay using ACS2 antibodies. (F) Ethylene production in response to ACC. Seedlings were treated with ACC in GC vials and ethylene in the headspace was measured 24–28h later. In the experiment, the ACC stock solution was prepared in water. For MG132 treatment, MG132 was dissolved in DMSO; an equal volume of DMSO was used as a control. The ACS6 immune complex activity was performed using specific anti-ACS6 antibodies. The results presented show average values of the triplicate experiment in three replicates each (n = 9). Error bars show standard error. Stars * indicate statistically significant difference at P < 0.05. Molecular Plant 2014 7, 960-976DOI: (10.1093/mp/ssu025) Copyright © 2014 The Authors. All rights reserved. Terms and Conditions

Figure 5 ABI1 Affects Ethylene Production, ACC Content, and ACS Activity during Ozone Stress. Arabidopsis thaliana plants were treated with 350 ppb of ozone for 6h. The results show a time course of ethylene production (A), ACC content (B), total ACS activity (C), ACS6, and ACS2 immune complex assay (D, E). The abi1td mutant shows a significant increase in ethylene production after 3h ozone treatment. An asterisk (*) indicates statistically significant changes at P < 0.05 from Student’s t-test. Data in are averages ± standard errors. Molecular Plant 2014 7, 960-976DOI: (10.1093/mp/ssu025) Copyright © 2014 The Authors. All rights reserved. Terms and Conditions

Figure 6 Ozone Tolerance of the abi1td Mutant. (A) Time course of ozone-induced leaf damage in abi1td and WT Col-0 plants. Plants were subjected to ozone treatment for 6h. The results are expressed as a percentage of the total ion content. The experiment was performed three times, with similar results, with at least five biological replicates each. The data show the means of 10–15 replicates. Data are averages ± standard errors. An asterisk (*) indicates statistically significant changes at P < 0.01 from Student’s t-test. (B–G) Increased total SOD and catalase activities in abi1td in response to ozone. Superoxide radical production (B), H2O2 content (C), and total SOD activity (D) in the abi1td mutant and the WT Col-0 plants response to ozone. (E) The abi1td mutant increased CSD2 protein level after 1h and 3h of ozone treatment. (F) APOX and catalase activities (G) in abi1td and the WT Col-0 plants. The asterisk (*) indicates statistically significant changes at P < 0.005. Molecular Plant 2014 7, 960-976DOI: (10.1093/mp/ssu025) Copyright © 2014 The Authors. All rights reserved. Terms and Conditions

Figure 7 Effect of Ozone on Ascorbate Pools in the abi1td Mutant. Levels (A, C, D) and redox state (B) of ascorbate in WT Col-0 and in the abi1td mutant. The bars represent mean ± SD (n = 6). AsA, ascorbate; FW, fresh weight. Data are averages ± standard errors. An asterisk (*) indicates statistically significant changes at P < 0.05; ** indicate statistically significant changes at P < 0.001. Molecular Plant 2014 7, 960-976DOI: (10.1093/mp/ssu025) Copyright © 2014 The Authors. All rights reserved. Terms and Conditions

Figure 8 Regulation of Ethylene Production by ABI1. ABI1 is involved in ethylene biosynthesis by affecting posttranscriptional regulation of type I ACS and by management of MPK6 activity. MPK6 positively regulates the stability of ACS2 and ACS6. The ABI1 protein phosphatase negatively regulates the activity of MPK6 and dephosphorylates ACS6. ABI1 also regulates the activity of enzymes involved in antioxidant defense system (Catalase, SOD, and APOX) and the level of ROS production in the cell. Oxidative stress induces the expression of the ABI1 gene and MPK6 activity. ABA inhibits ABI1 activity. Arrows indicate positive regulation, while flat-ended lines indicate negative regulation. Molecular Plant 2014 7, 960-976DOI: (10.1093/mp/ssu025) Copyright © 2014 The Authors. All rights reserved. Terms and Conditions