1. Volume 56, Issue 1, Pages 153-162 (October 2014)
Selective Protein Denitrosylation Activity of Thioredoxin-h5 Modulates Plant Immunity 
Sophie Kneeshaw, Silvère Gelineau, Yasuomi Tada, Gary J. Loake, Steven H. Spoel 
Molecular Cell 
Volume 56, Issue 1, Pages (October 2014)
DOI: /j.molcel
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2. Molecular Cell 2014 56, 153-162DOI: (10.1016/j.molcel.2014.08.003)
Copyright © 2014 Elsevier Inc. Terms and Conditions

3. Figure 1
The Plant TRX/NTR System Displays Protein Denitrosylation Activity
(A) BSA (20 μM) was S-nitrosylated with 1 mM GSNO and incubated with a TRX system consisting of TRXh5 (5 μM), NTRA (0.5 μM), and NADPH (1 mM). S-nitrosylated BSA (BSA-SNO) was detected using the Biotin-Switch Technique (BST) and is shown relative to total BSA. UV-induced SNO photolysis served as a control.
(B) Plant protein extracts were S-nitrosylated with 1 mM GSNO and incubated with the TRXh5/NTRA system as in (A). Protein-SNO were purified with the BST in presence or absence of ascorbate (Asc) and visualized by silver staining.
(C) WT protoplasts were incubated with 2 mM CysNO for the indicated times. Protein-SNO were purified using the BST and visualized by immunoblotting (IB) with an anti-biotin antibody.
(D– F) WT protoplasts were pretreated with or without 100 μM DNCB, followed by incubation with either 2 mM CysNO for 20 min (D), 2 mM GSNO for 10 min (E), or 2 mM DEA-NO for 10 min (F). Protein-SNO were detected by measuring formation of fluorescent triazolofluorescein (DAF-2T) and normalized against protein concentrations.
(G) WT, trx-h3 trx-h5 double, and ntra single mutant protoplasts were treated with 2 mM CysNO for 10 min. Protein-SNO were detected by measuring formation of fluorescent triazolofluorescein (DAF-2T) and normalized against protein concentrations.
Error bars in (D)–(G) represent SD (n = 3). See also Figure S1.
Molecular Cell  , DOI: ( /j.molcel )
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4. Figure 2
Trans-Denitrosylation Activity of TRXh5 Reverses SNO Modifications
(A) Incubation of oxymyoglobin (peaks at 542 and 580 nm) with GSNO and TRXh5 released NO⋅, converting oxymyoglobin into metmyoglobin (peak at 632 nm).
(B) Incubation of oxymyoglobin with GSNO and either TRXh5 or mutant TRXh5(C42S) released NO⋅, resulting in conversion of oxymyoglobin into metmyoglobin.
(C) BSA (20 μM) was S-nitrosylated with 1 mM GSNO and incubated with TRXh5 (40 μM). S-nitrosylated BSA (BSA-SNO) and TRXh5 (TRXh5-SNO) were detected with the BST and are shown relative to total BSA and TRXh5.
(D) BSA (20 μM) was S-nitrosylated with 1 mM GSNO and incubated with WT or an active site mutant (C39/C42S) of TRXh5 (40 μM). S-nitrosylated BSA (BSA-SNO) and TRXh5 (TRXh5-SNO) were detected with the BST and are shown relative to total BSA and TRXh5. UV-induced SNO photolysis served as a control.
(E) BSA (20 μM) was S-nitrosylated with 1 mM GSNO and incubated with WT or the active site mutants TRXh5(C39S) or TRXh5(C42S) (40 μM). S-nitrosylated BSA (BSA-SNO) and TRXh5 (TRXh5-SNO) were detected with the BST and are shown relative to total BSA and TRXh5. UV-induced SNO photolysis served as a control.
(F) Schematic of nondenaturing BST, utilizing immobilized mutant TRXh5(C42S). See text for details.
(G) Protein extracts were spiked with BSA and treated with or without 1 mM GSNO and subjected to the nondenaturing BST, as shown in (F). Purified denitrosylated plant proteins and BSA (deSNO-BSA) were detected using anti-biotin and anti-BSA antibodies, respectively, and are shown relative to total BSA.
(H) Plant protein extracts were treated with 1 mM GSNO and then incubated with immobilized His-tagged TRXh5(C42S) and TRXh5(C39/42S) mutants. Mixed disulphide intermediates formed by TRXh5 (TRXh5-substrate) were separated by non-reducing (−DTT) and reducing (+DTT) SDS-PAGE and detected using an anti-His antibody.
(I) Protoplasts from plants transformed with or without 35S::Myc-TRXh5 were treated with 2 mM GSNO for 10 min. Total protein was extracted under denaturing conditions and immunoprecipitated with an anti-Myc antibody. Mixed disulphide intermediates formed by TRXh5 (TRXh5-substrate) were separated by nonreducing (−DTT) and reducing (+DTT) SDS-PAGE and detected using an anti-Myc antibody.
See also Figure S2.
Molecular Cell  , DOI: ( /j.molcel )
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5. Figure 3
TRXh5 Selectively Restores Immune Deficiencies Caused by Elevated Protein-SNO
(A) WT, gsnor1, nox1, 35S::TRXh5 (gsnor1), and 35S::TRXh5 (nox1) plants were treated with 0.5 mM SA for the indicated times. The expression of TRXh5 was assessed by RT-PCR using gene-specific primers.
(B) Morphological phenotypes of 4-week-old WT, gsnor1, nox1, 35S::TRXh5 (gsnor1), and 35S::TRXh5 (nox1) plants.
(C) WT, gsnor1, and 35S::TRXh5 (gsnor1) plants were infected with Pst DC3000 (5 × 105 cells) and growth of Pst DC3000 was assessed after 5 days. Cfu, colony forming units. Error bars represent 95% confidence limits (n = 8). Asterisks indicate statistically significant differences compared to the WT (Tukey-Kramer ANOVA test; α = 0.05, n = 8).
(D) WT, nox1, and 35S::TRXh5 (nox1) plants were infected and analyzed as in (C).
(E) Extracts from plants expressing NPR1-GFP were incubated with either recombinant TRXh5 or the active site mutant TRXh5(C42S) together with a small amount of DTT (0.33 mM) to recycle TRX activity. Reduction of NPR1-GFP oligomer (O) to monomer (M) relative to total (T) NPR1-GFP was followed for the indicated times by non-reducing (−DTT) and reducing (+DTT) SDS-PAGE and detected using an anti-GFP antibody.
(F) WT, nox1, and 35S::TRXh5C42S (nox1) plants were infected and analyzed as in (C).
Molecular Cell  , DOI: ( /j.molcel )
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6. Figure 4 TRXh5 Selectively Denitrosylates Protein-SNO
(A) Mutant nox1 and gsnor1 plants with or without the 35S::Flag-TRXh5 transgene were treated with 0.5 mM SA. Total protein was extracted and incubated with or without the alkylating agent AMS, which prevents nonspecific disulphide formation. Proteins were separated by SDS-PAGE in the presence or absence of DTT and analyzed by western blotting using an anti-Flag antibody. Indicated are free TRXh5 monomer, mixed disulphide intermediates between TRXh5 and substrates (TRXh5-substrate), and total levels of TRXh5.
(B) Mutant nox1 and gsnor1 plants transformed with or without the 35S::Flag-TRXh5 transgene were infected with Pst DC3000 (5 × 106 cells). After 24 hr, SNO content was detected by measuring formation of fluorescent triazolofluorescein (DAF-2T), normalized against protein concentrations, and expressed as percentage of untransformed controls. Error bars represent SD (n = 3).
(C) Protoplasts were transformed with 35S::YFP-GSNOR1 or 35S::YFP-TRXh5 and subcellular localization analyzed by confocal microscopy.
Molecular Cell  , DOI: ( /j.molcel )
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7. Figure 5
TRXh5 Restores SA-Dependent Immune Signaling by Selective Protein Denitrosylation
(A) In-vitro-translated Flag-tagged NPR1 was S-nitrosylated with 1 mM GSNO and incubated with or without the TRX system, consisting of TRXh5 (5 μM), NTRA (0.5 μM), and NADPH (1 mM). S-nitrosylated NPR1 (NPR1-SNO) was detected with the BST in presence or absence of ascorbate (Asc) and is shown relative to total NPR1.
(B) Protoplasts from the indicated genotypes were transformed with 35S::NPR1-GFP and subcellular localization analyzed by confocal microscopy.
(C) WT, gsnor1, nox1, 35S::TRXh5 (gsnor1), and 35S::TRXh5 (nox1) plants were infected with Pst DC3000 (5 × 105 cells). After 24 hr expression of SA-dependent PR-1, WRKY38, and WRKY62, genes were analyzed using qPCR and normalized against constitutively expressed UBQ5. Error bars represent SD (n = 3).
(D) WT, gsnor1, nox1, 35S::TRXh5 (gsnor1), and 35S::TRXh5 (nox1) plants were treated with 0.5 mM SA for the indicated times. Expression of SA-dependent PR-1, WRKY38, and WRKY62 genes were analyzed using RT-PCR. Constitutively expressed UBQ10 was used as a loading control.
Molecular Cell  , DOI: ( /j.molcel )
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8. Figure 6
Proposed Model Showing that the SNO Reductases GSNOR1 and TRXh5 Regulate Different Branches of Protein-SNO in Plant Immune Signaling
Two classes of proteins are shown. Class Ι proteins are S-nitrosylated by GSNO, the level of which is regulated by the SNO reductase GSNOR1 (left panel). Class ΙΙ proteins are S-nitrosylated by free NO⋅ or other unknown intermediates and are denitrosylated by the TRXh5/NTRA system (right panel). As exemplified by the immune coactivator NPR1, class Ι and class ΙΙ proteins partly overlap and, consequently, are regulated by both GSNOR1 and the TRXh5/NTRA system (middle panel). Both pathways contribute to SA-dependent gene expression and immunity.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2014 Elsevier Inc. Terms and Conditions