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.

Acknowledgements

This research was funded by the National Natural Science Foundation of China (Grant Nos. 32260753, 31760582) and the Yunnan Ten Thousand Talents Plan: Young & Elite Talents Project.

Author contributions

H.X. designed the experiments and wrote the manuscript. S.Z., X.S., J.Z., and X.L. performed the experiments and analysed the data. X.S., P.G.-C., H.N., and K.L. helped in revising the manuscript.

Data availability

The whole of the data used in this study are available in the publication and its supplementary information files.

Conflict of interest statement

We declare the absence of any conflicts of interest.

Supplementary data

Supplementary data is available at Horticulture Research online.

References

[1] Tilman D, Cassman KG, Matson PA. et al. Agricultural sustainability and intensive production practices. Nature. 2002; 418: 671-7

[2] Ju XT, Kou CL, Christie P. et al. Changes in the soil environment from excessive application of fertilizers and manures to two contrasting intensive cropping systems on the North China plain. Environ Pollut. 2007; 145:497-506

[3] Yu L, Ma S, Zhang X. et al. Ancient rapid functional differentiation and fixation of the duplicated members in rice Dof genes after whole genome duplication. Plant J: Cell Mol Biol. 2021; 108: 1365-81

[4] Qin SQ, Quan Z, Ma J. et al. Regulating nitrate excess in lettuce-planted greenhouse soil with available carbon addition through irrigation. Environ Sci Pollut R. 2019; 26:19241-9

[5] Xu HN, He XZ, Wang K. et al. Identification of early nitrate stress response genes in spinach roots by suppression subtractive hybridization. Plant Mol Biol Report. 2012; 30:633-42

[6] Du J, Guo S, Sun J. et al. Proteomic and physiological analyses reveal the role of exogenous spermidine on cucumber roots in response to Ca(NO₃)₂ stress. Plant Mol Biol. 2018; 97:1-21

[7] Hossain Z, Pillai BV-S, Gruber MY. et al. Transcriptome profiling of Brassica napus stem sections in relation to differences in lignin content. BMC Genomics. 2018; 19:255

[8] Zhang Y, Chen H, Li S. et al. Comparative physiological and proteomic analyses reveal the mechanisms of Brassinolide-mediated tolerance to calcium nitrate stress in tomato. Front Plant Sci. 2021; 12:724288

[9] Kolbert Z, Barroso JB, Brouquisse R. et al. A forty year journey: the generation and roles of NO in plants. Nitric Oxide: Biol Chem. 2019; 93:53-70

[10] Kwon E, Feechan A, Yun B-W. et al. AtGSNOR1 function is required for multiple developmental programs in Arabidopsis. Planta. 2012; 236:887-900

[11] Yu MD, Lamattina L, Spoel SH. et al. Nitric oxide function in plant biology: a redox cue in deconvolution. New Phytol. 2014; 202: 1142-56

[12] Fancy NN, Bahlmann AK, Loake GJ. Nitric oxide function in plant abiotic stress. Plant Cell Environ. 2017; 40:462-72

[13] Astier J, Kulik A, Koen E. et al. Protein S-nitrosylation: what’s going on in plants? Free Radic Biol Med. 2012; 53: 1101-10

[14] Feng J, Chen LC, Zuo JR. Protein S-Nitrosylation in plants: current progresses and challenges. J Integr Plant Biol. 2019; 61: 1206-23

[15] Sircar E, Stoyanovsky DA, Billiar TR. et al. Analysis of glutathione mediated S-(de)nitrosylation in complex biological matrices by immuno-spin trapping and identification of two novel substrates. Nitric Oxide: Biol Chem. 2022; 118:26-30

[16] Corpas FJ, Barroso JB. Peroxynitrite (ONOO) is endogenously produced in arabidopsis peroxisomes and is overproduced under cadmium stress. Ann Bot-London. 2014; 113:87-96

[17] Rasool G, Buchholz G, Yasmin T. et al. Overexpression of SlGSNOR impairs in vitro shoot proliferation and developmental architecture in tomato but confers enhanced disease resistance. J Plant Physiol. 2021; 261:153433

[18] Feechan A, Kwon E, Yun BW. et al. A central role for S-nitrosothiols in plant disease resistance. Proc Natl Acad Sci USA. 2005; 102:8054-9

[19] Chen YY, Huang YF, Khoo KH. et al. Mass spectrometry-based analyses for identifying and characterizing S-nitrosylation of protein tyrosine phosphatases. Methods. 2007; 42:243-9

[20] Lee SJ, Lee JR, Kim YH. et al. Investigation of tyrosine nitration and nitrosylation of angiotensin II and bovine serum albumin with electrospray ionization mass spectrometry. Rapid Commun Mass Spectrom: RCM. 2007; 21:2797-804

[21] Shelar SB, Kaminska KK, Reddy SA. et al. Thioredoxin-dependent regulation of AIF-mediated DNA damage. Free Radic Biol Med. 2015; 87:125-36

[22] Li H, Wan A. Apoptosis of rheumatoid arthritis fibroblast-like synoviocytes: possible roles of nitric oxide and the thioredoxin 1. Mediat Inflamm. 2013; 2013:953462

[23] Haendeler J, Hoffmann J, Zeiher AM. Redox regulatory and anti-apoptotic functions of thioredoxin depend on S-nitrosylation at cysteine 69. Circulation. 2002; 106:212-3

[24] Bashandy T, Guilleminot J, Vernoux T. et al. Interplay between the NADP-linked thioredoxin and glutathione systems in Arabidopsis auxin signaling. Plant Cell. 2010; 22:376-91

[25] Calderon A, Sánchez-Guerrero A, Ortiz-Espín A. et al. Lack of mitochondrial thioredoxin o1 is compensated by antioxidant components under salinity in Arabidopsis thaliana plants. Physiol Plant. 2018; 164:251-67

[26] Sun LJ, Ren H, Liu R. et al. An h-type thioredoxin functions in tobacco defense responses to two species of viruses and an abiotic oxidative stress. Mol Plant Microbe In. 2010; 23:1470-85

[27] Wu FW, Nie L, Yu KN. et al. Characteristics of three thioredoxin genes and their role in chilling tolerance of harvested Banana fruit. Int J Mol Sci. 2016; 17:1526

[28] Ji MG, Park HJ, Cha J-Y. et al. Expression of Arabidopsis thaliana thioredoxin-h2 in Brassica napus enhances antioxidant defenses and improves salt tolerance. Plant Physiol Bioch. 2020; 147:313-21

[29] Salava H, Thula S, Mohan V. et al. Application of genome editing in tomato breeding: mechanisms, advances, and prospects. Int J Mol Sci. 2021; 22:682

[30] Chandrasekaran M, Boopathi T, Paramasivan M. A status-quo review on CRISPR-Cas9 gene editing applications in tomato. Int J Biol Macromol. 2021; 190:120-9

[31] García-Caparrós P. Breeding for yield quality parameters and abiotic stress in tomato using genome editing. In: Ricroch A. et al.eds., A Roadmap for Plant Genome Editing. Switzerland: Springer Nature, 2024,395-409

[32] Zhai J, Qi Q, Wang M. et al. Overexpression of tomato thioredoxin h (SlTrxh) enhances excess nitrate stress tolerance in transgenic tobacco interacting with SlPrx protein. Plant Sci. 2022; 315: 111137

[33] Ji R, Min J, Wang Y. et al. The role of plant growth regulators in modulating root architecture and tolerance to high-nitrate stress in tomato. Front Plant Sci. 2022; 13:864285

[34] Foyer CH, Noctor G. Redox homeostasis and antioxidant signaling: a metabolic interface between stress perception and physiological responses. Plant Cell. 2005; 17:1866-75

[35] Meyer AJ, Dreyer A, Ugalde JM. et al. Shifting paradigms and novel players in Cys-based redox regulation and ROS signaling in plants - and where to go next. Biol Chem. 2021; 402: 399-423

[36] Kaur H, Kaur H, Kaur H. et al. The beneficial roles of trace and ultratrace elements in plants. Plant Growth Regul. 2023; 100: 219-36

[37] Ali F, Li YH, Li FG. et al. Genome-wide characterization and expression analysis of cystathionine beta-synthase genes in plant development and abiotic stresses of cotton (Gossypium spp.). Int J Biol Macromol. 2021; 193:823-37

[38] Luan J, Dong J, Song X. et al. Overexpression of Tamarix hispida ThTrx5 confers salt tolerance to Arabidopsis by activating stress response signals. Int J Mol Sci. 2020; 21:1165

[39] Duan XH, Wang Z, Zhang Y. et al. Overexpression of a thioredoxin-protein-encoding gene, MsTRX, from Medicago sativa enhances salt tolerance to transgenic tobacco. Agronomy-Basel. 2022; 12:1467

[40] Gietler M, Nykiel M, Orzechowski S. et al. Proteomic analysis of S-nitrosylated and S-glutathionylated proteins in wheat seedlings with different dehydration tolerances. Plant Physiol Biochem : PPB. 2016; 108:507-18

[41] Daurelio LD, Romero MS, Petrocelli S. et al. Characterization of Citrus sinensis transcription factors closely associated with the non-host response to Xanthomonas campestris pv. Vesicatoria. J Plant Physiol. 2013; 170:934-42

[42] Hurali DT, Bhurta R, Tyagi S. et al. Analysis of NIA and GSNOR family genes and nitric oxide homeostasis in response to wheat-leaf rust interaction. Sci Rep. 2022; 12:803

[43] Kumar A, Dubey AK, Kumar V. et al. Over-expression of chickpea glutaredoxin (CaGrx) provides tolerance to heavy metals by reducing metal accumulation and improved physiological and antioxidant defence system. Ecotoxicol Environ Saf. 2020; 192:110252

[44] Kang BC, Wu Q, Sprague S. et al. Ectopic overexpression of an Arabidopsis monothiol glutaredoxin AtGRXS 17 affects floral development and improves response to heat stress in chrysanthemum (chrysanthemum morifolium Ramat.). Environ Exp Bot. 2019; 167:103864

[45] Ning X, Sun Y, Wang C. et al. A Rice CPYC-type Glutaredoxin OsGRX20 in protection against bacterial blight, methyl viologen and salt stresses. Front Plant Sci. 2018; 9:111

[46] Ströher E, Grassl J, Carrie C. et al. Glutaredoxin S15 is involved in Fe-S cluster transfer in mitochondria influencing lipoic acid-dependent enzymes, plant growth, and arsenic tolerance in Arabidopsis. Plant Physiol. 2016; 170:1284-99

[47] Wang M, Hao J, Chen X. et al. SlMYB102 expression enhances low-temperature stress resistance in tomato plants. PeerJ. 2020; 8:e10059

[48] Jahan MA, Harris B, Lowery M. et al. Glyceollin transcription factor GmMYB29A2 regulates soybean resistance to phytophthora sojae. Plant Physiol. 2020; 183:530-46

[49] Zhang X, Chen L, Shi Q. et al. SlMYB102, an R2R3-type MYB gene, confers salt tolerance in transgenic tomato. Plant Sci. 2020; 291:110356

[50] Liu ZY, Li XP, Zhang TQ. et al. Overexpression of ThMYB8 mediates salt stress tolerance by directly activating stress-responsive gene expression. Plant Sci. 2021; 302:110668

[51] Zhao K, Cheng Z, Guo Q. et al. Characterization of the poplar R2R3-MYB gene family and over-expression of PsnMYB108 confers salt tolerance in transgenic tobacco. Front Plant Sci. 2020; 11:571881

[52] Zhang P, Wang R, Yang X. et al. The R2R3-MYB transcription factor AtMYB49 modulates salt tolerance in Arabidopsis by modulating the cuticle formation and antioxidant defence. Plant Cell Environ. 2020; 43:1925-43

[53] Song Y, Yang W, Fan H. et al. TaMYB86B encodes a R2R3-type MYB transcription factor and enhances salt tolerance in wheat. Plant Sci. 2020; 300:110624

[54] Kim W-N, Kim H-J, Chung Y-S. et al. Construction of multiple guide RNAs in CRISPR/Cas9 vector using stepwise or simultaneous Golden Gate cloning: case study for targeting the FAD2 and FATB multigene in soybean. Plan Theory. 2021; 10:2542

[55] Zheng P, Bai XG, Long J. et al. Nitric oxide enhances the nitrate stress tolerance of spinach by scavenging ROS and RNS. Sci Hortic-Amsterdam. 2016; 213:24-33

[56] Kong FY, Deng Y, Zhou B. et al. A chloroplast-targeted DnaJ protein contributes to maintenance of photosystem II under chilling stress. J Exp Bot. 2014; 65:143-58

[57] Qi Q, Yanyan D, Yuanlin L. et al. Overexpression of SlMDHAR in transgenic tobacco increased salt stress tolerance involving S-nitrosylation regulation. Plant Sci: Int J Exp Plant Biol. 2020; 299:110609

[58] Rao KVM, Sresty TVS. Antioxidative parameters in the seedlings of pigeonpea (Cajanus cajan (L.) Millspaugh) in response to Zn and Ni stresses. Plant Sci. 2000; 157:113-28

[59] Gay CA, Gebicki JM. Measurement of protein and lipid hydroperoxides in biological systems by the ferric-xylenol orange method. Anal Biochem. 2003; 315:29-35

[60] Madhava Rao KV, Sresty TV. Antioxidative parameters in the seedlings of pigeonpea (Cajanus cajan (L.) Millspaugh) in response to Zn and Ni stresses. Plant Sci. 2000; 157: 113-28

[61] Cakmak I, Marschner H. Magnesium deficiency and high light intensity enhance activities of superoxide dismutase, ascorbate peroxidase, and glutathione reductase in bean leaves. Plant Phys-iol. 1992; 98:1222-7

[62] Mazel A, Leshem Y, Tiwari BS. et al. Induction of salt and osmotic stress tolerance by overexpression of an intracellular vesicle trafficking protein AtRab 7 (AtRabG3e). Plant Physiol. 2004; 134: 118-28

[63] Astier J, Besson-Bard A, Lamotte O. et al. Nitric oxide inhibits the ATPase activity of the chaperone-like AAA + ATPase CDC48, a target for S-nitrosylation in cryptogein signalling in tobacco cells. Biochem J. 2012; 447:249-60

[64] Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976; 72: 248-54

[65] Serpa V, Vernal J, Lamattina L. et al. Inhibition of AtMYB2 DNA-binding by nitric oxide involves cysteine S-nitrosylation. Biochem Biophys Res Commun. 2007; 361:1048-53

[66] Frungillo L, de Oliveira JF, Saviani EE. et al. Modulation of mitochondrial activity by S-nitrosoglutathione reductase in Arabidopsis thaliana transgenic cell lines. Biochim Biophys Acta. 2013; 1827: 239-47