1. Volume 67, Issue 4, Pages 702-710.e4 (August 2017)
Nitric Oxide Regulates Protein Methylation during Stress Responses in Plants 
Jiliang Hu, Huanjie Yang, Jinye Mu, Tiancong Lu, Juli Peng, Xian Deng, Zhaosheng Kong, Shilai Bao, Xiaofeng Cao, Jianru Zuo 
Molecular Cell 
Volume 67, Issue 4, Pages e4 (August 2017)
DOI: /j.molcel
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2. Molecular Cell 2017 67, 702-710.e4DOI: (10.1016/j.molcel.2017.06.031)
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3. Figure 1 Cys-125 of PRMT5 Is S-Nitrosylated
(A) Analysis of the S-nitrosylated His-PRMT5 recombinant protein by the biotin-switch assay. Purified His-PRMT5 recombinant protein was treated with GSNO or GSH (negative control) and then subjected to a biotin-switch assay. The S-nitrosylated protein was detected by an anti-biotin antibody.
(B) Analysis of S-nitrosylation of PRMT5 in vivo. Total protein extracts were prepared from wild-type plants and subjected to a biotin-switch assay. The S-nitrosylated protein was detected by an anti-PRMT5 antibody. Treatment without ascorbate sodium (Asc) is served as a negative control.
(C) Mass spectrometry analysis of the tryptic fragments of GSNO-treated His-PRMT5 recombinant protein. Cys-125 charged with biotin is identified as an S-nitrosylated residue.
(D) Analysis of the number of the S-nitrosylated residues in GSNO tripeptide (a standard containing a single S-nitrosylated Cys residue), His-PRMT5, and His-PRMT5C125S recombinant proteins treated with GSNO. The analysis was performed with the DAN-NAT assay (see STAR Methods).
(E) Analysis of the S-nitrosylation of His-PRMT5 and His-PRMT5C125S recombinant proteins by the biotin-switch assay. Quantification of the data is shown below the blot.
(F) Analysis of the S-nitrosylation of PRMT5-FLAG and PRMT5C125S-FLAG proteins by the biotin-switch assay. Quantification of the data is shown below the blot.
Error bars in (D)–(F) indicate SD of three independent experiments; two-tailed Student’s t test, ∗∗∗p < See also Figure S1 and Table S1.
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4. Figure 2
S-Nitrosylation at Cys-125 Enhances PRMT5 Methyltransferase Activity
(A) Analysis of the methylation of histone H4 catalyzed by His-PRMT5 and His-PRMT5C125S recombinant proteins treated with GSH (negative control) or GSNO or not treated (control).
(B) A quantitative analysis of the data shown in (A).
(C) Analysis of the methylation of myelin basic protein (MBP) catalyzed by His-PRMT5 and His-PRMT5C125S recombinant proteins treated with GSH (negative control) or GSNO or not treated (control).
(D) A quantitative analysis of the data shown in (C).
(E) Analysis of the methylation of SM-like protein (LSM4) catalyzed by His-PRMT5 and His-PRMT5C125S recombinant proteins treated with GSH (negative control) or GSNO or not treated (control).
(F) A quantitative analysis of the data shown in (E).
Error bars in (B), (D), and (F) indicate SD of three independent experiments; two-tailed Student’s t test, ∗∗p < See also Figure S2 and Table S1.
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5. Figure 3
S-Nitrosylation at Cys-125 Enhances Arg Symmetric Dimethylation and Salt Tolerance
(A) 5-day-old seedlings with the indicated genotypes germinated and grown on 1/2 MS agar plates containing 100 mM NaCl or 0.5 μM abscisic acid (ABA) as indicated. Scale bar, 0.5 cm.
(B) A quantitative analysis of the cotyledon greening rate of seedlings shown in (A) (n = 100).
(C) Analysis of the primary root length of seedlings with the indicated genotypes. 4-day-old seedlings germinated and grown on 1/2 MS medium were transferred onto 1/2 MS medium with or without (control) 100 mM NaCl and cultured for an additional 7 days. Photos were taken at day 11. Scale bar, 1 cm.
(D) A quantitative analysis of the primary root length shown in (C) (n = 30).
(E) Immunoblotting analysis of Arg symmetric dimethylation using an SYM11 antibody. Total protein extracts were prepared from 11-day-old seedlings with the indicated genotypes treated with or without 200 mM NaCl for 6 hr and then used for the immunoblotting analysis.
(F) A quantitative analysis of the signal intensity of the indicated bands (red arrow) shown in (E). Data presented are the average values of three independent experiments.
(G) Analysis of pre-mRNA splicing of AT1G18160 by RT-PCR. Seedlings with the indicated genotypes were grown on 1/2 MS medium for 11 days and then treated with 200 mM NaCl for 6 hr. Total RNA was prepared and then used for RT-PCR. The aberrantly spliced RNA band is indicated by an arrow. The experiments were performed at least three times with similar results.
Error bars in (B), (D), and (F) indicate SD; two-tailed Student’s t test; ∗∗p < 0.01, ∗∗∗p < See also Figures S1 and S3 and Table S1.
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6. Figure 4
Nitric Oxide Positively Regulates Arg Symmetric Dimethylation and Salt Tolerance by S-Nitrosylation of PRMT5
(A) Biotin-switch analysis of the S-nitrosylated PRMT5 in wild-type seedlings treated with or without 200 mM NaCl for 6 hr. A quantitative analysis of S-nitrosylated PRMT5 is shown below the blot.
(B) Analysis of the S-nitrosylated PRMT5 in Col-0 and gsnor1-3 seedlings. A quantitative analysis of S-nitrosylated PRMT5 is shown below the blot.
(C) Immunoblotting analysis of Arg symmetric dimethylation using an SYM11 antibody. Total protein extracts were prepared from 11-day-old seedlings with the indicated genotypes treated with or without 200 mM NaCl for 6 hr and then used for the immunoblotting analysis.
(D) A quantitative analysis of the signal intensity of the indicated bands (red arrow) shown in (C).
(E) A quantitative analysis of relative fresh weight (weight of NaCl treated/control) of plants (n = 30). The relative fresh weight of gsnor1-3 seedlings is set at 1.0.
(F) 5-day-old seedlings with the indicated genotypes germinated and grown on 1/2 MS agar plates containing various combinations of 150 mM NaCl and 50 μM GSNO as indicated. Scale bar, 1 cm.
(G) A quantitative analysis of the cotyledon greening rate of seedlings shown in (F) (n = 100).
(H) 7-day-old seedlings with the indicated genotypes germinated and grown on 1/2 MS agar plates containing various combinations of 1 μM ABA and 50 μM GSNO as indicated. Scale bar, 1 cm.
(I) A quantitative analysis of the cotyledon greening rate of seedlings shown in (H) (n = 100).
Error bars in (A), (B), (D), (E), (G), and (I) indicate SD of three independent experiments (biological replicates); two-tailed Student’s t test; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p <  See also Figure S4 and Table S1.
Molecular Cell  , e4DOI: ( /j.molcel )
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