1. Volume 11, Issue 7, Pages 970-982 (July 2018)
The Antagonistic Action of Abscisic Acid and Cytokinin Signaling Mediates Drought Stress Response in Arabidopsis 
Xiaozhen Huang, Lingyan Hou, Jingjing Meng, Huiwen You, Zhen Li, Zhizhong Gong, Shuhua Yang, Yiting Shi 
Molecular Plant 
Volume 11, Issue 7, Pages (July 2018)
DOI: /j.molp
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2. Figure 1 ARR5 Positively Regulates ABA Response through SnRK2s.
(A) ARR5 protein levels in 35S:HF-ARR5 and ARR5:ARR5-Myc transgenic plants. Total proteins were extracted from 10-day-old seedlings. Anti-HA antibody was used to detect ARR5 protein. Actin was used as a loading control.
(B and C) Germination phenotype (B) and greening rates (C) of Col-0, 35S:HF-ARR5, and ARR5:ARR5-Myc seedlings grown on MS medium with or without 0.5 μM ABA at 22°C for 10 days.
(D and E) Drought tolerance phenotype (D) and survival rate (E) of Col-0, 35S:HF-ARR5, and ARR5:ARR5-Myc seedlings in soil before treatment (top) or on day 3 after rewatering following drought treatment for 10 days (bottom).
(F) qRT-PCR showed that the expression level of ABA-responsive genes (ABI1, ABI5, RD29B, and RAB18) in wild-type Col-0 and 35S:HF-ARR5 plants. 10-day-old seedlings were treated with 50 μM ABA for 0 h or 3 h. The relative expression levels of ABA-responsive genes in untreated Col were set to 1.
(G and H) Germination phenotype (G) and greening rate (H) of wild-type Col-0, HF-ARR5, snrk2.2,2.3, and snrk2.2,2.3 HF-ARR5 seedlings grown on the MS medium with or without 0.5 μM ABA at 22°C for 7 days.
(I and J)Phenotype (I) and survival rate (J) of wild-type Col, snrk2.6, snrk2.6 HF-ARR5, and HF-ARR5 plants after drought treatment. Nine plants in each pot in the same tray were watered and grown on soil at 22°C for 10 days before the photograph was taken (left of I). The plants were then subjected to water-deficit stress for 7 days, followed by rewatering. The photograph was taken at day 3 after rewatering (right of I). The survival rate was scored.
In (C), (E), (H) and (J), data are means of three biological replicates ±SD (n = 60 for C and H, n = 27 for E and J), and the asterisks indicate a significant difference between the mutants and wild-type Col receiving the same treatment. *P < 0.05, **P < 0.01, ***P < 0.001 (Student t-test).
Molecular Plant  , DOI: ( /j.molp )
Copyright © 2018 The Author Terms and Conditions

3. Figure 2 Subgroup III SnRK2s Interact with ARR5 In Vitro and In Vivo.
(A) BiFC analysis showing the interaction of ARR5 and SnRK2.3/2.6 in N. benthamiana. ARR5-YFPN was co-transformed into N. benthamiana leaves with YFPC or SnRK2.3/2.6-YFPC.
(B) LCI assay showing the interaction between ARR5 and SnRK2.2/2.3/2.6. ARR5-cLuc or cLuc was co-transformed into N. benthamiana leaves with nLuc or SnRK2.2/2.3/2.6-nLuc.
(C) Yeast two-hybrid analysis showing the interaction between ARR5 and SnRK2.6. Yeast cells were plated on selection media: SD-LW (-Leu/-Trp) and SD-LWHA (-Leu/-Trp/-His/-Ade).
(D) LCI assay showing the interaction between ARR5-cLuc and SnRK2.6-nLuc in tobacco leaves.
Molecular Plant  , DOI: ( /j.molp )
Copyright © 2018 The Author Terms and Conditions

4. Figure 3 SnRK2s Phosphorylate ARR5 In Vitro and In Vivo.
(A) In vitro kinase assay of ARR5 phosphorylation by SnRK2.2, 2.3, and 2.6. Phosphorylation of ARR5 by SnRK2.6 (left) and by SnRK2.2 and SnRK2.3 (right) is shown (top). Coomassie brilliant blue (CBB) staining was used as a loading control (bottom). The exposure time was about 30 s for SnRK2.6 and 3 min for SnRK2.2 and 2.3. The bands were quantified using ImageJ.
(B) SnRK2.6 in-gel kinase assay showed ARR5 is phorsphorylated by SnRK2.6 in vivo. 10-day-old wild-type Col-0 and snrk2.6 mutant seedlings were treated with 50 μM ABA or a mock treatment for 0.5 h, and total proteins were separated on an SDS–PAGE gel containing 3 mg/ml ARR5-His protein, followed by incubation with kinase buffer in the presence of [γ-32P]ATP. An autoradiograph of the gel is shown on the top, with arrowheads indicating the ABA-induced bands representing ARR5-His protein phosphorylated by activated SnRK2.6, and asterisks indicating non-specific bands. Immunoblotting with anti-SnRK2.6 and anti-HSP90 antibodies was done as a loading control.
(C) In vitro kinase assay showed phosphorylation of wild type and mutated forms ARR5 by SnRK2.6. Potential phosphorylated Ser residues of ARR5 were mutated to Ala (ARR5m); ARR5S21A, S48A, S72A, S117A is abbreviated as ARR54A (bottom). Purified ARR5-His or ARR5m-His fusion proteins were phosphorylated by SnRK2.6 before LC–MS. The relative fold change in the amount of protein for each ARR5 mutation band was calculated using ImageJ software and normalized to the level of ARR5-His. CBB staining was done as a loading control.
(D) Immunoblot analysis with anti-HA antibody showing the upshift of ARR5 and ARR54A in a phos-tag gel. The upshift of ARR5 is abolished after treatment with λPPase. Total proteins were extracted from 10-day-old seedlings and subjected to immunoblot analysis. ARR5 or ARR54A and HSP90 proteins separated on an SDS–PAGE gel without phos-tag were detected as loading controls.
Molecular Plant  , DOI: ( /j.molp )
Copyright © 2018 The Author Terms and Conditions

5. Figure 4 SnRK2s Phosphorylate ARR5 to Promote Its Protein Stability.
(A) ARR5 protein stability in the presence of ABA. 10-day-old 35S:HF-ARR5 seedlings (upper panel) were treated with 300 μM CHX for 2 h, followed by treatment with DMSO, 50 μM ABA, or 50 μM MG132 for the indicated amount of time.
(B) The levels of HF-ARR5 protein in the snrk2.2,2.3 or snrk2.6 background. 10-day-old HF-ARR5, snrk2.2,2.3 HF-ARR5, and snrk2.6 HF-ARR5 seedlings were grown on MS with or without 0.5 μM ABA.
(C) The level of HF-ARR5 and HF-ARR54A proteins. 10-day-old HF-ARR5 and HF-ARR54A seedlings were grown on MS.
(D) The stability of the HF-ARR5 and HF-ARR54A proteins. 10-day-old HF-ARR5 and HF-ARR54A seedlings were treated with 300 μM CHX for 0, 3, and 6 h. DMSO treatment was used as a mock control.
In (A) to (D), anti-HA antibody was used to determine the level of ARR5 protein. Actin or HSP90 was used as a loading control. Similar results were obtained in three independent experiments.
(E and F) Germination phenotype (E) and greening rate (F) of wild-type Col-0, 35S:HF-ARR5 and 35S:HF-ARR54A seedlings grown on MS medium with or without 0.5 μM ABA at 22°C for 10 days.
(G and H) Primary root phenotype (G) and primary root length (H) of Col-0, 35S:HF-ARR5, and 35S:HF-ARR54A. 5-day-old seedlings grown vertically on MS medium were transferred to MS medium supplemented with or without 20 μM ABA for 10 days. Scale bar, 1 cm.
(I) Expression of ABA-responsive genes in wild-type Col and 35S:HF-ARR5, HF-ARR54A, and arr5 plants. 10-day-old seedlings were treated with 50 μM ABA for 0 h or 3 h. The relative expression levels of ABA-responsive genes in Col-0 were set to 1.
In (F), (H), and (I), data are means of three biological replicates ±SD (n = 60 for F, and n = 30 for H). *P < 0.05, **P < 0.01, ***P < (Student t-test).
Molecular Plant  , DOI: ( /j.molp )
Copyright © 2018 The Author Terms and Conditions

6. Figure 5
ARR1, ARR11 and ARR12 Interact with Subgroup III SnRK2s and Inhibit Their Kinase Activities.
(A and B) ARR1 (A) and ARR11 (B) interact with SnRK2s in coIP assays. ARR1-Flag or ARR11-Flag and SnRK2-Myc constructs were co-transformed into Arabidopsis protoplasts. Total protein extracts were immunoprecipitated with anti-Myc beads, and the proteins from crude lysates (input) and immunoprecipitated proteins were detected with anti-Myc and anti- Flag antibodies.
(C) ARR12 interacts with SnRK2s in the BiFC assay. ARR12-YFPN or YFPN was co-transformed into N. benthamiana leaves with SnRK2.2-YFPC, SnRK2.3-YFPC, or SnRK2.6-YFPC.
(D) ARR1, ARR11 and ARR12 inhibit the kinase activity of SnRK2s in vitro. The asterisks indicate bands corresponding to phosphorylated ARR1 and ARR11. Coomassie brilliant blue staining (CBB) was used as the loading control.
(E–G) In-gel kinase assay showed SnRK2.6 activity in wild-type and cytokinin signaling mutants after treatment with 50 μM ABA or mock treatment for 0.5 h. Truncated recombinant ABF2 was used as the substrate. The arrowheads indicate the ABA-induced bands representing phosphorylation of ABF2 by activated SnRK2.6. The asterisks indicate non-specific bands. The relative fold change in intensity of ABA-inducible bands in the mutants after ABA treatment was calculated and normalized to the Col-0 controls. Immunoblot analysis with anti-SnRK2.6 and anti-HSP90 antibodies was done as the loading control. Three independent experiments were performed with similar results.
Molecular Plant  , DOI: ( /j.molp )
Copyright © 2018 The Author Terms and Conditions

7. Figure 6 Genetic Analysis of SnRK2s and ARR1/11/12.
(A and B) Germination phenotype (A) and greening rate (B) of Col, snrk2.2,2.3, arr1,11,12, and snrk2.2,2.3 arr1,11,12 seedlings grown on MS medium with or without 0.5 μM ABA for 7 days at 22°C.
(C and D) Primary root phenotype (C) and root length (D) of Col-0, snrk2.2,2.3 arr1,11,12, and snrk2.2,2.3 arr1,11,12 seedlings. Five-day-old seedlings grown vertically on MS medium were transferred to MS medium supplemented with or without 20 μM ABA for 10 days. Scale bar, 1 cm.
(E) Expression levels of ABA-responsive genes in Col-0, snrk2.2,2.3, snrk2.2,2.3 arr1,11,12, and arr1,11,12. Ten-day-old seedlings were treated with 50 μM ABA for 0 h or 3 h. The relative fold change in gene expression in the mutants after ABA treatment was calculated and normalized to the expression levels in the untreated Col control.
(F and G) Phenotype (F) and survival rate (G) of Col-0, snrk2.6, arr1,11,12, snrk2.6 arr1,11,12 seedlings in soil before treatment (left) or 3 days after re-watering following drought treatment for 10 days (right).
In (B), (D), (E) and (G), data are means of three biological replicates ±SD (n = 60 for B, n = 30 for D, and n = 27 for G). **P < 0.01, ***P < 0.001 (Student t-test).
Molecular Plant  , DOI: ( /j.molp )
Copyright © 2018 The Author Terms and Conditions

8. Figure 7
Working Model for SnRK2-ARR Interactions during Drought Stress Responses.
Under non-stressed conditions, cytokinin activates cytokinin signaling and antagonizes ABA signaling by inhibiting SnRK2 activity via the type-B ARRs (ARR1, ARR11, and ARR12), and thus promotes plant growth and development. Upon drought stress, ABA-activated SnRK2s phosphorylate the type-A ARR5 and enhance its stability, thus amplifying the ABA-mediated stress response. Meanwhile, the type-A ARR5 represses cytokinin signaling via a negative feedback loop to restrict plant growth. Solid and dashed lines indicate direct and indirect regulation, respectively.
Molecular Plant  , DOI: ( /j.molp )
Copyright © 2018 The Author Terms and Conditions