Thioredoxin h2 inhibits the MPKK5-MPK3 cascade to regulate the CBF–COR signaling pathway in Citrullus lanatus suffering chilling stress

Abstract

Thioredoxins (TRXs) are ubiquitous oxidoreductases and present as a multigenic family. TRXs determine the thiol redox balance, which is crucial for plants in the response to cold stress. However, limited knowledge is available about the role of TRXs in watermelon ( Citrullus lanatus), which is highly sensitive to chilling stress in agricultural practice. Here, we identified 18 genes encoding 14 typical and 4 atypical TRXs from the watermelon genome, and found that ClTRX h2 localized at the plasma membrane was largely induced by chilling. Virus-induced gene silencing of ClTRX h2 resulted in watermelon plants that were more sensitive to chilling stress. We further found that ClTRX h2 physically interacted with mitogen-activated protein kinase kinase 5 (ClMPKK5), which was confirmed to phosphorylate and activate ClMPK3 in vitro, and the activation of ClMPK3 by ClMPKK5 was blocked by a point mutation of the Cys-229 residue to Ser in ClMPKK5. Additionally, ClTRX h2 inhibited the chilling-induced activation of ClMPK3, suggesting that the ClMPKK5–ClMPK3 cascade is regulated in a redox-dependent manner. We showed that ClMPK3-silenced plants had increased tolerance to chilling, as well as enhanced transcript abundances of the C-repeat/DREB binding factor ( ClCBF) and cold-responsive ( ClCOR) genes. Taken together, our results indicate that redox status mediated by ClTRX h2 inhibits ClMPK3 phosphorylation through the interaction between ClTRX h2 and ClMPKK5, which subsequently regulates the CBF–COR signaling pathway when submitted to chilling stress. Hence, our results provide a link between thiol redox balance and MAPK cascade signaling, revealing a conceptual framework to understand how TRX regulates chilling stress tolerance in watermelon.

Acknowledgements

This research was funded by the National Natural Science Foundation of China (32172551, 32002046), the Fundamental Research Funds for the Central Universities (2662020YLPY003), the China Agriculture Research System of MOF and MARA (CARS-25), and the Natural Science Foundation of Hubei Province (2019CFA017). Dr Qinsheng Gu from Zhengzhou Fruit Research Institute, Chinese Academy of Agricultural Sciences, provided the pV190 vector and watermelon seeds (Zhengkang 2).

Author contributions

F.C. designed the research; A.Q.X., N.N.W., H.H., and S.Z. conducted the experiments; A.Q.X. and F.C. analyzed the data and wrote the manuscript; Y.H., Q.S.K., and Z.L.B. supervised the study; W.F.N provided intellectual support and all the authors revised the manuscript.

Data availability

All the supplementary data relevant to this study are presented online.

Conflict of interest

The authors declare that they have no conflicts of interest.

References

[1] Considine MJ, Foyer CH . Redox regulation of plant development. Antioxidants Redox Signaling. 2014; 21: 1305-26.

[2] Dietz KJ . Redox signal integration: from stimulus to networks and genes. Physiol Plant. 2008; 133: 459-68.

[3] Buchanan BB . The path to thioredoxin and redox regulation in chloroplasts. Annu Rev Plant Biol. 2016; 67: 1-24.

[4] Lee S, Kim SM, Lee RT . Thioredoxin and thioredoxin target proteins: from molecular mechanisms to functional significance. Antioxid Redox Signal. 2013; 18: 1165-207.

[5] Lu J, Holmgren A . Thioredoxin system in cell death progression. Antioxid Redox Signal. 2012; 17: 1738-47.

[6] Shu N, Hagglund P, Cai H et al. Modification of Cys residues in human thioredoxin-1 by p-benzoquinone causes inhibition of its catalytic activity and activation of the ASK1/p38-MAPK signalling pathway. Redox Biol. 2020; 29: 101400.

[7] Niso-Santano M, Gonzalez-Polo RA, Bravo-San Pedro JM et al. Activation of apoptosis signal-regulating kinase 1 is a key factor in paraquat-induced cell death: modulation by the Nrf2/Trx axis. Free Radic Biol Med. 2010; 48: 1370-81.

[8] Yeo EJ, Shin MJ, Yeo HJ et al. Tat-thioredoxin 1 reduces inflammation by inhibiting pro-inflammatory cytokines and modulating MAPK signaling. Exp Ther Med. 2021; 22: 1395.

[9] Chibani K, Pucker B, Dietz KJ et al. Genome-wide analysis and transcriptional regulation of the typical and atypical thioredoxins in Arabidopsis thaliana . FEBS Lett. 2021; 595: 2715-30.

[10] Meyer Y, Reichheld JP, Vignols F . Thioredoxins in Arabidopsis and other plants . Photosynth Res. 2005; 86: 419-33.

[11] Chibani K, Wingsle G, Jacquot J-P et al. Comparative genomic study of the thioredoxin family in photosynthetic organisms with emphasis on Populus trichocarpa . Mol Plant. 2009; 2: 308-22.

[12] Chibani K, Tarrago L, Gualberto JM et al. Atypical thioredoxins in poplar: the glutathione-dependent thioredoxin-like 2.1 supports the activity of target enzymes possessing a single redox active cysteine. Plant Physiol. 2012; 159: 592-605.

[13] Laloi C, Rayapuram N, Chartier Y et al. Identification and characterization of a mitochondrial thioredoxin system in plants. Proc Natl Acad Sci USA. 2001; 98: 14144-9.

[14] Gelhaye E, Rouhier N, Gerard J et al. A specific form of thioredoxin h occurs in plant mitochondria and regulates the alternative oxidase. Proc Natl Acad Sci USA. 2004; 101: 14545-50.

[15] Renard M, Alkhalfioui F, Schmitt-Keichinger C et al. Identification and characterization of thioredoxin h isoforms differentially expressed in germinating seeds of the model legume Medicago truncatula . Plant Physiol. 2011; 155: 1113-26.

[16] Hagglund P, Finnie C, Yano H et al. Seed thioredoxin h. Biochim Biophys Acta. 2016; 1864: 974-82.

[17] Olate E, Jimenez-Gomez JM, Holuigue L et al. NPR1 mediates a novel regulatory pathway in cold acclimation by interacting with HSFA1 factors. Nature Plants. 2018; 4: 811-23.

[18] Lee ES, Park JH, Wi SD et al. Redox-dependent structural switch and CBF activation confer freezing tolerance in plants. Nature Plants. 2021; 7: 914-22.

[19] Xie G, Kato H, Sasaki K et al. A cold-induced thioredoxin h of rice, OsTrx23, negatively regulates kinase activities of OsMPK3 and OsMPK6 in vitro. FEBS Lett. 2009; 583: 2734-8.

[20] Barrero-Gil J, Salinas J . Post-translational regulation of cold acclimation response. Plant Sci. 2013; 205-206: 48-54.

[21] Shi Y, Ding Y, Yang S . Molecular regulation of CBF signaling in cold acclimation. Trends in Plant Sci. 2018; 23: 623-37.

[22] Li H, Ding Y, Shi Y et al. MPK3-and MPK6-mediated ICE1 phosphorylation negatively regulates ICE1 stability and freezing tolerance in Arabidopsis . Dev Cell. 2017; 43: 630-642.e4.

[23] Zhao C, Wang P, Si T et al. MAP kinase cascades regulate the cold response by modulating ICE1 protein stability. Dev Cell. 2017; 43: 618-629.e5.

[24] Gelhaye E, Rouhier N, Jacquot JP . The thioredoxin h system of higher plants. Plant Physiol Biochem. 2004; 42: 265-71.

[25] Ying Y, Yue W, Wang S et al. Two h-type thioredoxins interact with the E2 ubiquitin conjugase PHO2 to fine-tune phosphate homeostasis in rice. Plant Physiol. 2017; 173: 812-24.

[26] Park JH, Lee ES, Chae HB et al. Disulfide reductase activity of thioredoxin-h2 imparts cold tolerance in Arabidopsis . Biochem Biophys Res Commun. 2021; 568: 124-30.

[27] Kovtun Y, Chiu WL, Tena G et al. Functional analysis of oxidative stress-activated mitogen-activated protein kinase cascade in plants. Proc Natl Acad Sci USA. 2000; 97: 2940-5.

[28] Meng J, Fu L, Liu K et al. Global profiling of distinct cysteine redox forms reveals wide-ranging redox regulation in C. elegans . Nature Commun. 2021; 12: 1415.

[29] Hourihan JM, Mazzeo LEM, Fernandez-Cardenas LP et al. Cysteine sulfenylation directs IRE-1 to activate the SKN-1/Nrf2 antioxidant response. Mol Cell. 2016; 63: 553-66.

[30] Ren D, Yang H, Zhang S . Cell death mediated by MAPK is associated with hydrogen peroxide production in Arabidopsis . J Biol Chem. 2002; 277: 559-65.

[31] Mittler R, Vanderauwera S, Gollery M et al. Reactive oxygen gene network of plants. Trends Plant Sci. 2004; 9: 490-8.

[32] Dos Santos CV, Rey P . Plant thioredoxins are key actors in the oxidative stress response. Trends Plant Sci. 2006; 11: 329-34.

[33] Balmer Y, Koller A, del Val G et al. Proteomics gives insight into the regulatory function of chloroplast thioredoxins. Proc Natl Acad Sci USA. 2003; 100: 370-5.

[34] Balmer Y, Vensel WH, Tanaka CK et al. Thioredoxin links redox to the regulation of fundamental processes of plant mitochondria. Proc Natl Acad Sci USA. 2004; 101: 2642-7.

[35] Yamazaki D, Motohashi K, Kasama T et al. Target proteins of the cytosolic thioredoxins in Arabidopsis thaliana . Plant Cell Physiol. 2004; 45: 18-27.

[36] Vanacker H, Guichard M, Bohrer AS et al. Redox regulation of monodehydroascorbate reductase by thioredoxin y in plastids revealed in the context of water stress. Antioxidants. 2018; 7: 183.

[37] Elasad M, Ahmad A, Wang H et al. Overexpression of CDSP32 (GhTRX134) cotton gene enhances drought, salt, and oxidative stress tolerance in Arabidopsis . Plants (Basel). 2020; 9: 1388.

[38] 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.

[39] Irazoqui JE, Urbach JM, Ausubel FM . Evolution of host innate defence: insights from Caenorhabditis elegans and primitive invertebrates . Nat Rev Immunol. 2010; 10: 47-58.

[40] Xie G, Sasaki K, Imai R et al. A redox-sensitive cysteine residue regulates the kinase activities of OsMPK3 and OsMPK6 in vitro. Plant Sci. 2014; 227: 69-75.

[41] Kundumani-Sridharan V, Subramani J, Das KC . Thioredoxin activates MKK4-NF κB pathway in a redox-dependent manner to control manganese superoxide dismutase gene expression in endothelial cells . J Biol Chem. 2015; 290: 17505-19.

[42] Nakamura H, Nakamura K, Yodoi J . Redox regulation of cellular activation. Annu Rev Immunol. 1997; 15: 351-69.

[43] Tobiume K, Matsuzawa A, Takahashi T et al. ASK1 is required for sustained activations of JNK/p38 MAP kinases and apoptosis. EMBO Rep. 2001; 2: 222-8.

[44] Agarwal PK, Agarwal P, Reddy MK et al. Role of DREB transcription factors in abiotic and biotic stress tolerance in plants. Plant Cell Rep. 2006; 25: 1263-74.

[45] Mizoi J, Shinozaki K, Yamaguchi-Shinozaki K . AP2/ERF family transcription factors in plant abiotic stress responses. Biochim Biophys Acta. 2012; 1819: 86-96.

[46] Cheng F, Lu J, Gao M et al. Redox signaling and CBF-responsive pathway are involved in salicylic acid-improved photosynthesis and growth under chilling stress in watermelon. Front Plant Sci. 2016; 7: 1519.

[47] Jaglo-Ottosen KR, Gilmour SJ, Zarka DG et al. Arabidopsis CBF1 overexpression induces COR genes and enhances freezing tolerance . Science. 1998; 280: 104-6.

[48] Vogel JT, Zarka DG, Van Buskirk HA et al. Roles of the CBF2 and ZAT12 transcription factors in configuring the low temperature transcriptome of Arabidopsis . Plant J. 2005; 41: 195-211.

[49] Ito Y, Katsura K, Maruyama K et al. Functional analysis of rice DREB1/CBF-type transcription factors involved in cold-responsive gene expression in transgenic rice. Plant Cell Physiol. 2006; 47: 141-53.

[50] Jaglo KR, Kleff S, Amundsen KL et al. Components of the Arabidopsis C-repeat/dehydration-responsive element binding factor cold-response pathway are conserved in Brassica napus and other plant species . Plant Physiol. 2001; 127: 910-7.

[51] Gilmour SJ, Zarka DG, Stockinger EJ et al. Low temperature regulation of the Arabidopsis CBF family of AP2 transcriptional activators as an early step in cold-induced COR gene expression . Plant J. 1998; 16: 433-42.

[52] Haake V, Cook D, Riechmann JL et al. Transcription factor CBF4 is a regulator of drought adaptation in Arabidopsis . Plant Physiol. 2002; 130: 639-48.

[53] Chinnusamy V, Ohta M, Kanrar S et al. ICE1: a regulator of cold-induced transcriptome and freezing tolerance in Arabidopsis . Genes Dev. 2003; 17: 1043-54.

[54] Kim YS, Lee M, Lee JH et al. The unified ICE-CBF pathway provides a transcriptional feedback control of freezing tolerance during cold acclimation in Arabidopsis . Plant Mol Biol. 2015; 89: 187-201.

[55] Zhang Z, Li J, Li F et al. OsMAPK3 phosphorylates OsbHLH002/OsICE1 and inhibits its ubiquitination to activate OsTPP1 and enhances rice chilling tolerance. Dev Cell. 2017; 43: 731-743.e5.

[56] Liu M, Liang Z, Aranda MA et al. A cucumber green mottle mosaic virus vector for virus-induced gene silencing in cucurbit plants. Plant Methods. 2020; 16: 9.

[57] Hodges DM, DeLong JM, Forney CF et al. Improving the thiobarbituric acid-reactive-substances assay for estimating lipid peroxidation in plant tissues containing anthocyanin and other interfering compounds. Planta. 1999; 207: 604-11.

[58] Sheludko YV, Sindarovska YR, Gerasymenko IM et al. Comparison of several Nicotiana species as hosts for high-scale Agrobacterium-mediated transient expression . Biotechnol Bioeng. 2007; 96: 608-14.

[59] Chen H, Zou Y, Shang Y et al. Firefly luciferase complementation imaging assay for protein-protein interactions in plants. Plant Physiol. 2008; 146: 368-76.

[60] Kong Q, Yuan J, Gao L et al. Evaluation of appropriate reference genes for gene expression normalization during watermelon fruit development. PLoS One. 2015; 10: e0130865.

[61] Livak KJ, Schmittgen TD . Analysis of relative gene expression data using real-time quantitative PCR and the 2 ^−ΔΔCT method . Methods. 2001; 25: 402-8.