SlTrxh functions downstream of SlMYB86 and positively regulates nitrate stress tolerance via S-nitrosation in tomato seedling

Abstract

Nitric oxide (NO) is a redox-dependent signaling molecule that plays a crucial role in regulating a wide range of biological processes in plants. It functions by post-translationally modifying proteins, primarily through S-nitrosation. Thioredoxin (Trx), a small and ubiquitous protein with multifunctional properties, plays a pivotal role in the antioxidant defense system. However, the regulatory mechanism governing the response of tomato Trxh (SlTrxh) to excessive nitrate stress remains unknown. In this study, overexpression or silencing of SlTrxh in tomato led to increased or decreased nitrate stress tolerance, respectively. The overexpression of SlTrxh resulted in a reduction in levels of reactive oxygen species (ROS) and an increase in S-nitrosothiol (SNO) contents; conversely, silencing SlTrxh exhibited the opposite trend. The level of S-nitrosated SlTrxh was increased and decreased in SlTrxh overexpression and RNAi plants after nitrate treatment, respectively. SlTrxh was found to be susceptible to S-nitrosation both in vivo and in vitro, with Cysteine 54 potentially being the key site for S-nitrosation. Protein interaction assays revealed that SlTrxh physically interacts with SlGrx9, and this interaction is strengthened by S-nitrosation. Moreover, a combination of yeast one-hybrid (Y1H), electrophoretic mobility shift assay (EMSA), chromatin immunoprecipitation-quantitative PCR (ChIP-qPCR), and transient expression assays confirmed the direct binding of SlMYB86 to the SlTrxh promoter, thereby enhancing its expression. SlMYB86 is located in the nucleus and SlMYB86 overexpressed and knockout tomato lines showed enhanced and decreased nitrate stress tolerance, respectively. Our findings indicate that SlTrxh functions downstream of SlMYB86 and highlight the potential significance of S-nitrosation of SlTrxh in modulating its function under nitrate stress.

Figure 1

SlTrxh positively regulates the excess nitrate stress tolerance in tomato. a Photographs of SlTrxh overexpression (OE) and RNAi lines grown on wet filter paper with or without excess nitrate. The scale bar represents 4 cm. b Root length of plants in a. c Plant height of tomatoes in a. d Fresh weight of tomatoes in a. e Photographs of 2-week-old SlTrxh OE and RNAi plants after 20 days of excess nitrate treatment. f Root length of seedlings in e. g Plant height of seedlings in e. h Fresh weight of seedlings in e. i Quantitative RT-PCR analysis of SlTrxh expression in WT and SlTrxh transgenic plants. j SlTrxh protein level in WT, OE, and RNAi plants. The quantification of the data, carried out using the ImageJ software, is shown below the blot. The lowercase letters a, b, c, and d are used to indicate significant variations among different groups or conditions at a significance level of P < 0.05. Data represent the mean ± SE (n = 3).

Figure 2

Reactive oxygen species scavenging capacity of SlTrxh overexpression and RNAi tomato plants under excess nitrate stress. The tomato seedlings, aged two weeks, were subjected to irrigation with either a 10 mM solution of nitrate (control) or a 100 mM nitrate solution for a period of 20 days. a ROS dye H₂DCFDA staining of primary root tips. Scale bar is 200 μm. b The relative fluorescence intensity of a was carried out using the ImageJ software. c MDA contents. d H₂O₂ and O₂^·− histochemical staining with DAB (left) and NBT (right). e–l activities of antioxidant enzymes of SOD (e), CAT (f), APX (g) and expression of antioxidant enzymes of SlSOD (h), SlCAT (i), SlAPX (j), SlNTRB (k), SlTPX (l) in tomato leaves. Data represent the mean ± SE (n = 3).

Figure 3

S-nitrosation is important for the function of SlTrxh under nitrate stress. The tomato seedlings, aged two weeks, were subjected to irrigation with either a 10 mM solution of nitrate (control) or a 100 mM nitrate solution for a period of 20 days. a The impact of excessive nitrate stress on the accumulation of NO—NO dye DAF-FM staining of primary root tips. Scale bar is 200 μm. NO accumulation was done with 10 μM DAF-FM in 10 mM Tris–HCl in the root tips of the main root. b The relative fluorescence intensity of a was carried out using the ImageJ software. c SNOs contents. d  SlNR mRNA transcript level. e S-nitrosated SlTrxh level in SlTrxh overexpression and RNAi transgenic tomato seedlings. The quantification of the data, which was carried out using the ImageJ software, is shown below the blot. Data represent the mean ± SE (n = 3).

Figure 4

S-nitrosation analysis of SlTrxh in vivo and in vitro. a S-nitrosated levels of SlTrxh in tomato leaves treated with excess nitrate. Tomato seedlings were treated with 10 mM of nitrate (CK) or 100 mM of nitrate for 24 hours. 100 ng total protein of tomato leaves were loaded per lane and subjected for Biotin Switch Test (BST). b 10 ng purified recombinant Trxh protein was treated with increased concentrations of GSNO (250, 500, 1000, 2000 μM GSNO, 500 μM GSH, or 500 μM DTT) and underwent BST. GSH and DTT were served as negative controls. Quantification of the data is shown below the blot. c Mass spectrometric analyses identified Cys54 as the S-nitrosated site in the SlTrxh protein. The MS/MS spectra of Cys54 originated from a biotin-charged SlTrxh peptide (NTICKPPAVGK). d Effects of Cys 54, 98, and 101 to Ser site-directed mutation on S-nitrosation of SlTrxh upon GSNO treatment. 10 ng of recombinant SlTrxh protein were loaded per lane. Quantification of the data is shown below the blot. e S-nitrosation of SlTrxh in SlTrxh^C54S overexpressed transgenic tobacco. #1, #2, and #3 refer to different tobacco transgenic lines in which the amino acid Cys54 of SlTrxh has been substituted with Ser. 100 ng total protein of tobacco leaves were loaded per lane. Quantification of the data is shown below the blot.

Figure 5

SlTrxh physically interacts with SlGrx9. a Y2H assay showing interactions of SlTrxh and SlGrx9. Yeast cells with pGBKT7-SlGrx9 and pGADT7-SlTrxh were grown well both on SD/−Leu/−Trp and SD/−Leu/−Trp/-His/−Ade mediums. Positive control containing pGADT7-T + pGBKT7-53 and pGADT7-SlTrxh + BD, AD + pGBKT7-SlGrx9 and AD + BD were served as negative controls. b Co-IP assay showing SlTrxh and SlGrx9 interaction. SlTrxh and SlGrx9 were tagged with the GFP and Flag tags. The precipitate was detected using anti-GFP and anti-Flag antibodies. c Biomolecular luciferase complimentary assay (LCA) showing the interaction of SlTrxh and SlGrx9. d and e S-nitrosation enhances the interaction between SlTrxh and SlGrx9. The 54th cysteine residue of SlTrxh mutated into tryptophan (which is proposed to undergo nitrosation) or serine (which is proposed to lose nitrosation). Nitrate and GSNO were added in the LCA experiment. GSNO acted as a NO donor and cPTIO was used as a NO scavenger. The relative quantification was carried out using the ImageJ software.

Figure 6

SlMYB86 directly activates the expression of SlTrxh. a–b Electrophoretic mobility shift assay (EMSA) performed to investigate the binding of SlMYB86 to the promoter region of SlTrxh, which contains the CAACTG/TAACTG motif. c Yeast one-hybrid (Y1H) system of SlMYB86 binding to SlTrxh promoter. d Chromatin immunoprecipitation-quantitative PCR (ChIP-qPCR) assay of the enrichment of SlMYB86 activating SlTrxh promoter. Here, C0 was served as a negative control. e A schematic diagram of the dual-luciferase reporter sassy with reporter and effector vector. f Dual-luciferase reporter assay. g Relative quantification of f was carried out using the ImageJ software. h Dual-luciferase reporter of the transcription level of SlMYB86 on SlTrxh with nitrate. i Relative quantification of f.

Figure 7

SlMYB86 exhibits a positive role under nitrate stress conditions. a Subcellular localization of SlMYB86 in tobacco leaf epidermal cells. b Expression of SlMYB86 in tomato root under nitrate stress by qRT-PCR. c Phenotype of SlMYB86 overexpression transgenic tomato seedlings under nitrate stress. Scale bar is 4 cm. d–f Root length, plant height ,and fresh weight of OE and KO tomato seedlings under nitrate stress. g The expression level of SlTrxh in OE and KO plants of SlMYB86 under nitrate stress. Data represent the mean ± SE (n = 3).

Figure 8

A schematic illustration of SlTrxh positively regulates nitrate stress tolerance through S-nitrosation in tomato. In the SlTrxh overexpression plants, SlMYB86 directly binds to the promoter of SlTrxh, thereby activating its expression. The NO accumulation and the S-nitrosation of SlTrxh were induced under nitrate stress, with Cys54 identified as a crucial site for this process. The S-nitrosation enhances the interaction between SlTrxh and SlGrx9, thereby improving tomato’s resistance to nitrate stress with lower ROS accumulation. While in the SlTrxh RNAi plants, the expression and S-nitrosated level of SlTrxh was reduced and the interaction of SlTrxh and SlGrx9 was lower than overexpression plants under nitrate stress with more ROS accumulation.