Nitric oxide influences auxin signaling through S-nitrosylation of the Arabidopsis TRANSPORT INHIBITOR RESPONSE 1 auxin receptor

Abstract

Previous studies have demonstrated that auxin (indole-3-acetic acid) and nitric oxide (NO) are plant growth regulators that coordinate several plant physiological responses determining root architecture. Nonetheless, the way in which these factors interact to affect these growth and developmental processes is not well understood. The Arabidopsis thaliana F-box proteins TRANSPORT INHIBITOR RESPONSE 1/AUXIN SIGNALING F-BOX (TIR1/AFB) are auxin receptors that mediate degradation of AUXIN/INDOLE-3-ACETIC ACID (Aux/IAA) repressors to induce auxin-regulated responses. A broad spectrum of NO-mediated protein modifications are known in eukaryotic cells. Here, we provide evidence that NO donors increase auxin-dependent gene expression while NO depletion blocks Aux/IAA protein degradation. NO also enhances TIR1-Aux/IAA interaction as evidenced by pull-down and two-hybrid assays. In addition, we provide evidence for NO-mediated modulation of auxin signaling through S-nitrosylation of the TIR1 auxin receptor. S-nitrosylation of cysteine is a redox-based post-translational modification that contributes to the complexity of the cellular proteome. We show that TIR1 C140 is a critical residue for TIR1-Aux/IAA interaction and TIR1 function. These results suggest that TIR1 S-nitrosylation enhances TIR1-Aux/IAA interaction, facilitating Aux/IAA degradation and subsequently promoting activation of gene expression. Our findings underline the importance of NO in phytohormone signaling pathways.

Figure 1. IAA increases endogenous NO in Arabidopsis roots

Wild-type seedlings grown on ATS media were pre-loaded with the probe DAF-FM DA, and exposed to 1 μM IAA for 1.5 h at room temperature in darkness. NO accumulation is shown as green fluorescence in representative roots. Bar = 100 μM

Figure 2. NO is required for IAA-dependent gene expression and Aux/IAA protein degradation

(a) Arabidopsis BA3:GUS seedlings were treated with SNP with or without 10 nM IAA, and (b) pretreated for 45 min with cPTIO (0.5 mM) or hemoglobin (50 μM) prior 50 nM IAA treatment and them subjected to GUS staining (c) Wild-type seedlings were treated with different concentrations of IAA with or without increasing concentrations of SNP. Total RNA was isolated and transcript levels of IAA1 and IAA5 were analyzed using specific ³²P labeled probes. rRNA is shown as loading control. (d) HS:AXR3NT-GUS seedlings were treated with GSNO with or without 50 nM IAA or (e) with 1 μM IAA in combination with cPTIO (1 mM) or hemoglobin (50 μM) and stained for GUS activity.

Figure 3. Conservation of C140 and C480 in TIR1/AFB auxin receptor family and location in TIR1 crystal structure

(a) Alignment of amino acid sequences surrounding C140 and C480 in TIR1/AFB receptor family using ClustalX program. Asterisks stand for the cysteine residues. (b) Two views of the TIR1 structure shown as a ribbon diagram. Carbon alphas of C140 (red) and C480 (blue) are represented by spheres. IP6 (colored by element) and the IAA7 substrate peptide (yellow) molecules are shown as stick models. IAA is represented by a space filling model (green).

Figure 4. S-nitrosylation of TIR1 recombinant protein

Purified TIR1 protein was incubated with GSNO or GSH for 30 min and subjected to biotin-switch assay. S-nitrosylated TIR1 was detected with an anti-biotin antibody (upper panel). As a negative control GSNO-treated TIR1 was incubated with 20 mM DTT before the biotin-switch. Silver-stained TIR1 is shown as loading control.

Figure 5. NO enhances TIR1/AFB2-Aux/IAA interaction

Pull-down reactions were performed using ³⁵S-methionine in vitro synthesized (a) TIR1-Myc or (b) AFB2-Myc and recombinant GST-IAA3 proteins. Reactions were carried out in the presence of the indicated IAA concentrations and the addition of 1mM CysNO or 1 mM cPTIO. TIR1/AFB2 proteins were detected using Phosphoimager. Coomassie blue-stained GST-IAA3 was used as loading control. (c) Yeast two-hybrid assays were carried out with cells co-transformed with the indicated constructs and grown on SD-U-H-T selective media plus the addition of 50 μM IAA or increasing concentrations of SNP and X-Gal to develop β-galactosydase activity.

Figure 6. TIR1-Aux/IAA interaction is dependent on C140 and C480 of TIR1

(a) tir1-Myc C140A and tir1-Myc C480A mutated proteins were ³⁵S-methionine in vitro synthesized and used in pull-down reactions along recombinant GST-IAA3 proteins. Reactions were carried out in the presence or absence of 50 μM IAA and TIR1-Myc and mutant proteins were detected using Phosphoimager. Coomassie blue-stained GST-IAA3 was used as loading control. (b) Yeast two-hybrid assays were carried out with cells co-transformed with the indicated constructs and grown on SD-U-H-T selective media plus the addition of 50 μM IAA. (c) Mutations in amino acids surrounding C140 do not affect TIR1-Aux/IAA interaction. TIR1-Myc mutated versions on the S-nitrosylation consensus motif around C140 were used to carry out pull-down reactions. Experiments were performed as (a).

Figure 7

tir1 C140A overexpression does not rescue the tir1-1 auxin resistant phenotype. Four-day-old seedlings grown on ATS media were transferred to media containing 2,4-D. (a) Representative seedlings after 6 days under 2,4-D treatment. (b) Root elongation was measured after 2 days in 2,4-D treatment. (c) Lateral root formation was measured after 4 days in 2,4-D treatment. Values represent the mean of at least three independent experiments ± SE.