Tomato NAC2-DREB2 module fine-tunes saline–alkali stress sensitivity via modulation of melatonin biosynthesis and ROS homeostasis

Abstract

Soil salinization poses a serious threat to plant development and represents a major obstacle to the sustainable production of crops worldwide. Melatonin (MT) contributes prominently to plant tolerance against abiotic environments. However, the molecular basis of transcriptional regulation underlying melatonin accumulation in tomato under saline–alkali stress is still largely unknown. Herein, we identify SlNAC2, a NAC transcription factor in tomato induced by saline–alkali stress, which suppresses the key melatonin biosynthetic genes SlCOMT2 and SlSNAT, while activating SlCV, a gene linked to reactive oxygen species (ROS) accumulation and programmed cell death. These regulatory effects reduce MT levels and promote excessive ROS production, ultimately altering the plant’s tolerance to saline–alkali stress. Silencing of SlNAC2 through the RNA interference method significantly improves saline–alkali tolerance in tomato, while its constitutive overexpression shows increased susceptibility to saline–alkali stress. Further evidence reveals that under saline–alkali conditions, SlNAC2 directly targets cis-elements of SlCOMT2 and SlSNAT promoters, suppressing their transcription and consequently reducing melatonin levels, whereas simultaneously binding to the SlCV promoter to activate its expression, ultimately leading to ROS accumulation. Moreover, comprehensive protein interaction analyses confirmed that SlNAC2 physically associates with SlDREB2, a DREB-type transcription factor involved in salt stress response. Through its interaction with SlNAC2, SlDREB2 partially attenuates its repression of SlCOMT2 and SlSNAT, thereby increasing melatonin accumulation and ROS scavenging, ultimately enhancing tomato’s resilience to saline–alkali stress conditions. Collectively, our findings reveal a SlNAC2–SlDREB2 regulatory module that finely tunes melatonin synthesis and ROS levels to regulate tomato’s response to saline–alkali stress, providing new strategies for developing stress-resilient tomato varieties.

Acknowledgements

This work was supported by the Key R&D Program of Shandong Province (2022LZGCQY001), the Shandong Agriculture Research System (SDARS-05), Qingdao Science and Technology Public Welfare Demonstration Special Project (25-1-5-xdny-13-nsh and 25-1-5-xdny-2-nsh), and the National Natural Science Foundation of China (31701063, 31770275, 32472750, and U22A20495).

Author contributions

S.C.L., Y.S., S.L., A.W., and H.W. designed the experiments. S.C.L., Y.S., X.Z., W.Z., X.L., and F.W. performed experiments and analyzed data. S.C.L., Y.S., S.L., and H.W. wrote the manuscript. All authors have read and agreed to the published version of the manuscript.

Data availability

The data underlying this article are available in the article and in its online supplementary material.

Conflicts of interest statement

The authors have no conflicts of interest to declare.

Supplementary material

Supplementary material is available at Horticulture Research online.

References

[1] Cao Y, Zhang M, Liang X, et al. Natural variation of an EF-hand Ca ²⁺-binding-protein coding gene confers saline-alkaline tolerance in maize . Nat Commun. 2020; 11: 186.

[2] Zhang H, Yu F, Xie P, et al. A Gγ protein regulates alkaline sensitivity in crops. Science. 2023; 379: eade8416.

[3] Wang J, Zhang Y, Wang J, et al. Promoting γ-aminobutyric acid accumulation to enhances saline-alkali tolerance in tomato. Plant Physiol. 2024; 196: 2089-104.

[4] Xu W, Jia L, Shi W, et al. The tomato 14-3-3 protein TFT4 modulates H ⁺ efflux, basipetal auxin transport, and the PKS5-J3 pathway in the root growth response to alkaline stress . Plant Physiol. 2013; 163: 1817-28.

[5] Javid M, Ford R, Nicolas ME . Tolerance responses of Brassica juncea to salinity, alkalinity and alkaline salinity. Funct Plant Biol. 2012; 39: 699.

[6] Kaur R, Zhawar VK . Regulation of secondary antioxidants and carbohydrates by gamma-aminobutyric acid under salinity-alkalinity stress in rice (Oryza sativa L.). Biol Futur. 2021; 72: 229-39.

[7] VanWallendael A, Soltani A, Emery NC, et al. A molecular view of plant local adaptation: incorporating stress-response networks. Annu Rev Plant Biol. 2019; 70: 559-83.

[8] Fuglsang AT, Guo Y, Cuin TA, et al. Arabidopsis protein kinase PKS5 inhibits the plasma membrane H+-ATPase by preventing interaction with 14-3-3 protein. Plant Cell. 2007; 19: 1617-34.

[9] Yang Y, Guo Y . Unraveling salt stress signaling in plants. J Integr Plant Biol. 2018; 60: 796-804.

[10] Zhou H, Shi H, Yang Y, et al. Insights into plant salt stress signaling and tolerance. J Genet Genomics. 2024; 51: 16-34.

[11] Kim B, Waadt R, Cheong YH, et al. The calcium sensor CBL10 mediates salt tolerance by regulating ion homeostasis in Arabidopsis. Plant J. 2007; 52: 473-84.

[12] Tang R, Yang Y, Yang L, et al. Poplar calcineurin B-like proteins PtCBL10A and PtCBL10B regulate shoot salt tolerance through interaction with PtSOS2 in the vacuolar membrane. Plant Cell Environ. 2014; 37: 573-88.

[13] Lin H, Yang Y, Quan R, et al. Phosphorylation of SOS3-LIKE CALCIUM BINDING PROTEIN8 by SOS2 protein kinase stabilizes their protein complex and regulates salt tolerance in Arabidopsis. Plant Cell. 2009; 21: 1607-19.

[14] Egea I, Pineda B, Ortíz-Atienza A, et al. The SlCBL10 calcineurin B-like protein ensures plant growth under salt stress by regulating Na ⁺ and Ca ²⁺ homeostasis . Plant Physiol. 2018; 176: 1676-93.

[15] Wang L, Chen H, Chen G, et al. Transcription factor SlWRKY50 enhances cold tolerance in tomato by activating the jasmonic acid signaling. Plant Physiol. 2024; 194: 1075-90.

[16] Chaudhry UK, Gökçe ZNÖ, Gökçe AF . The influence of salinity stress on plants and their molecular mechanisms. Biol Life Sci Forum. 2022; 11: 31.

[17] Lu S, Sun Y, Liu X, et al. The SlbHLH92 transcription factor enhances salt stress resilience by fine-tuning hydrogen sulfide biosynthesis in tomato. Int J Biol Macromol. 2024; 282: 137294.

[18] Han K, Zhao Y, Sun Y, et al. NACs, generalist in plant life. Plant Biotechnol J. 2023; 21: 2433-57.

[19] Zhang X, Long Y, Huang J, et al. OsNAC45 is involved in ABA response and salt tolerance in rice. Rice. 2020; 13: 79.

[20] Xiong H, He H, Chang Y, et al. Multiple roles of NAC transcription factors in plant development and stress responses. J Integr Plant Biol. 2025; 67: 510-38.

[21] Chen X, Lu S, Wang Y, et al. OsNAC2 encoding a NAC transcription factor that affects plant height through mediating the gibberellic acid pathway in rice. Plant J. 2015; 82: 302-14.

[22] Mao C, Ding J, Zhang B, et al. OsNAC2 positively affects salt-induced cell death and binds to the OsAP37 and OsCOX11 promoters. Plant J. 2018; 94: 454-68.

[23] Mao C, Lu S, Lv B, et al. A Rice NAC transcription factor promotes leaf senescence via ABA biosynthesis. Plant Physiol. 2017; 174: 1747-63.

[24] Xi D, Chen X, Wang Y, et al. Arabidopsis ANAC092 regulates auxin-mediated root development by binding to the ARF8 and PIN4 promoters. J Integr Plant Biol. 2019; 61: 1015-31.

[25] Durian G, Sedaghatmehr M, Matallana-Ramirez LP, et al. Calcium-dependent protein kinase CPK1 controls cell death by in vivo phosphorylation of senescence master regulator ORE1. Plant Cell. 2020; 32: 1610-25.

[26] Sun Z, Wu M, Wang S, et al. An insertion of transposon in DcNAP inverted its function in the ethylene pathway to delay petal senescence in carnation (Dianthus caryophyllus L.). Plant Biotechnol J. 2023; 21: 2307- 21.

[27] Wang T, Ma X, Chen Y, et al. SlNAC3 suppresses cold tolerance in tomatoes by enhancing ethylene biosynthesis. Plant Cell Environ. 2024; 47: 3132-46.

[28] Ma F, Liang Y, Meng F, et al. The LbNAM2-LbZDS module enhances drought resistance in wolfberry (Lycium barbarum) by participating in ABA biosynthesis. Plant J. 2025; 121: e70077.

[29] Alshareef NO, Wang JY, Ali S, et al. Overexpression of the NAC transcription factor JUNGBRUNNEN1 (JUB1) increases salinity tolerance in tomato. Plant Physiol Biochem. 2019; 140: 113-21.

[30] Hong Y, Zhang H, Huang L, et al. Overexpression of a stress-responsive NAC transcription factor gene ONAC022 improves drought and salt tolerance in rice. Front Plant Sci. 2016; 7: 7.

[31] Mao C, Ding W, Wu Y, et al. Overexpression of a NAC-domain protein promotes shoot branching in rice. New Phytol. 2007; 176: 288-98.

[32] Zhong Q, Yu J, Wu Y, et al. Rice transcription factor OsNAC2 maintains the homeostasis of immune responses to bacterial blight. Plant Physiol. 2024; 195: 785-98.

[33] Shen J, Lv B, Luo L, et al. The NAC-type transcription factor OsNAC2 regulates ABA-dependent genes and abiotic stress tolerance in rice. Sci Rep. 2017; 7: 40641.

[34] Lu S, Sun Y, Wang Z, et al. Alfalfa transcription factor MsNAC2a orchestrates the homeostasis of salt stress responses via reactive oxygen species accumulation and hydrogen sulphide depletion. Plant Biotechnol J. 2025; 23: 5297-320.

[35] Zhuang J, Cai B, Peng R-H, et al. Genome-wide analysis of the AP2/ERF gene family in Populus trichocarpa. Biochem Biophys Res Commun. 2008; 371: 468-74.

[36] Dietz K-J, Vogel MO, Viehhauser A . AP2/EREBP transcription factors are part of gene regulatory networks and integrate metabolic, hormonal and environmental signals in stress acclimation and retrograde signalling. Protoplasma. 2010; 245: 3-14.

[37] Li T, Huang Y, Khadr A, et al. DcDREB1A, a DREB-binding transcription factor from Daucus carota, enhances drought tolerance in transgenic Arabidopsis thaliana and modulates lignin levels by regulating lignin-biosynthesis-related genes. Environ Exp Bot. 2020; 169: 103896.

[38] Chen Q, Li N, Cui X, et al. AP2/ERF transcription factors regulate the biosynthesis of terpenoids, phenolics, and alkaloids in plants. Hortic Res. 2025; 13: 280.

[39] Liu Q, Kasuga M, Sakuma Y, et al. Two transcription factors, DREB1 and DREB2, with an EREBP/AP2 DNA binding domain separate two cellular signal transduction pathways in drought- and low-temperature-responsive gene expression, respectively, in Arabidopsis. Plant Cell. 1998; 10: 1391-406.

[40] Yamaguchi-Shinozaki K, Shinozaki K . Transcriptional regulatory networks in cellular responses and tolerance to dehydration and cold stresses. Annu Rev Plant Biol. 2006; 57: 781-803.

[41] Peng Y, Cui L, Wang Y, et al. Pumpkin CmoDREB2A enhances salt tolerance of grafted cucumber through interaction with CmoNAC1 to regulate H2O2 and ABA signaling and K ⁺/Na ⁺ homeostasis . Hortic Res. 2024; 11: uhae057.

[42] Srivastava R, Kumar R . The expanding roles of APETALA2/ethylene responsive factors and their potential applications in crop improvement. Brief Funct Genomics. 2019; 18: 240-54.

[43] Lata C, Prasad M . Role of DREBs in regulation of abiotic stress responses in plants. J Exp Bot. 2011; 62: 4731-48.

[44] Mei F, Chen B, Du L, et al. A gain-of-function allele of a DREB transcription factor gene ameliorates drought tolerance in wheat. Plant Cell. 2022; 34: 4472-94.

[45] Thirumalaikumar VP, Devkar V, Mehterov N, et al. NAC transcription factor JUNGBRUNNEN1 enhances drought tolerance in tomato. Plant Biotechnol J. 2018; 16: 354-66.

[46] Gadjev I, Vanderauwera S, Gechev TS, et al. Transcriptomic footprints disclose specificity of reactive oxygen species signaling in Arabidopsis. Plant Physiol. 2006; 141: 436-45.

[47] Sakuma Y, Maruyama K, Osakabe Y, et al. Functional analysis of an Arabidopsis transcription factor, DREB2A, involved in drought-responsive gene expression. Plant Cell. 2006; 18: 1292-309.

[48] Suzuki N, Sejima H, Tam R, et al. Identification of the MBF1 heat-response regulon of Arabidopsis thaliana. Plant J. 2011; 66: 844-51.

[49] Hu X, Liang J, Wang W, et al. Comprehensive genome-wide analysis of the DREB gene family in Moso bamboo Phyllostachys edulis: evidence for the role of PeDREB28 in plant abiotic stress response. Plant J. 2023; 116: 1248-70.

[50] Hichri I, Muhovski Y, Clippe A, et al. SlDREB2, a tomato dehydration-responsive element-binding 2 transcription factor, mediates salt stress tolerance in tomato and Arabidopsis. Plant Cell Environ. 2016; 39: 62-79.

[51] Wang T, Yang J, Cao J, et al. MsbZIP55 regulates salinity tolerance by modulating melatonin biosynthesis in alfalfa. Plant Biotechnol J. 2025; 23: 2125-39.

[52] Zhang Z, Zhang Y . Melatonin in plants: what we know and what we don’t. Food Qual Saf. 2021; 5: fyab009.

[53] Arnao MB, Hernández-Ruiz J . Functions of melatonin in plants: a review. J Pineal Res. 2015; 59: 133-50.

[54] Zhang Z, Zhang X, Chen Y, et al. Understanding the mechanism of red light-induced melatonin biosynthesis facilitates the engineering of melatonin-enriched tomatoes. Nat Commun. 2023; 14: 5525.

[55] Wang C, Bian C, Li J, et al. Melatonin promotes Al ³⁺ compartmentalization via H ⁺ transport and ion gradients in Malus hupehensis . Plant Physiol. 2023; 193: 821-39.

[56] Yu J-C, Lu J-Z, Cui X-Y, et al. Melatonin mediates reactive oxygen species homeostasis via SlCV to regulate leaf senescence in tomato plants. J Pineal Res. 2022; 73: e12810.

[57] Liu G, Hu Q, Zhang X, et al. Melatonin biosynthesis and signal transduction in plants in response to environmental conditions. J Exp Bot. 2022; 73: 5818-27.

[58] Zhao D, Yao Z, Zhang J, et al. Melatonin synthesis genes N-acetylserotonin methyltransferases evolved into caffeic acid O-methyltransferases and both assisted in plant terrestrialization. J Pineal Res. 2021; 71: e12737.

[59] Gao Y, Chen H, Chen D, et al. Genetic and evolutionary dissection of melatonin response signaling facilitates the regulation of plant growth and stress responses. J Pineal Res. 2023; 74: e12850.

[60] Wei J-W, Liu M, Zhao D, et al. Melatonin confers saline-alkali tolerance in tomato by alleviating nitrosative damage and S-nitrosylation of H ⁺-ATPase 2 . Plant Cell. 2025; 37: koaf035.

[61] Guo Y, Li J, Liu L, et al. A self-amplifying NO-H2S loop mediates melatonin-induced CBF-responsive pathway and cold tolerance in watermelon. Plant J. 2025; 121: e70025.

[62] Zhu J, Zhang Y, Wang Y, et al. The ABF4-bHLH28-COMT5 module regulates melatonin synthesis and root development for drought tolerance in citrus. Plant J. 2025; 121: e70078.

[63] Shan Q, Zhao D, Cao B, et al. Jasmonic acid and nitric oxide orchestrate a hierarchical melatonin cascade for Botrytis cinerea resistance in tomato. Plant Physiol. 2025; 197: kiaf078.

[64] Ma J, Li C, Sun L, et al. The SlWRKY57-SlVQ21/SlVQ16 module regulates salt stress in tomato. J Integr Plant Biol. 2023; 65: 2437-55.

[65] Chen S, Zhang W, Zhang Q, et al. SlNAC12, a novel NAC-type transcription factor, confers salt stress tolerance in tomato. Plant Cell Rep. 2024; 44: 5.

[66] Wang G, Xu X, Wang H, et al. A tomato transcription factor, SlDREB3 enhances the tolerance to chilling in transgenic tomato. Plant Physiol Biochem. 2019; 142: 254-62.

[67] Wei L, Ren X, Qin L, et al. TaWRKY55-TaPLATZ2 module negatively regulate saline-alkali stress tolerance in wheat. J Integr Plant Biol. 2025; 67: 19-34.

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

[69] Zhang Q, Cai W, Ji T-T, et al. WRKY13 enhances cadmium tolerance by promoting D-CYSTEINE DESULFHYDRASE and hydrogen sulfide production. Plant Physiol. 2020; 183: 345-57.

[70] Sun Y, Song K, Guo M, et al. A NAC transcription factor from ‘Sea Rice 86’ enhances salt tolerance by promoting hydrogen sulfide production in rice seedlings. Int J Mol Sci. 2022; 23: 6435.

[71] Mao C, He J, Liu L, et al. OsNAC2 integrates auxin and cytokinin pathways to modulate rice root development. Plant Biotechnol J. 2020; 18: 429-42.

[72] Chen H, Chen X, Chen D, et al. A comparison of the low temperature transcriptomes of two tomato genotypes that differ in freezing tolerance: Solanum lycopersicum and Solanum habrochaites. BMC Plant Biol. 2015; 15: 132.

[73] Barrero-Gil J, Huertas R, Rambla JL, et al. Tomato plants increase their tolerance to low temperature in a chilling acclimation process entailing comprehensive transcriptional and metabolic adjustments. Plant Cell Environ. 2016; 39: 2303-18.

[74] Chen X, Mo F, Shen C, et al. Genome-wide identification and expression analysis of the SlNAC gene family in tomato based on a high-quality genome. Hortic Environ Biotechnol. 2022; 63: 887-901.

[75] Borgohain P, Saha B, Agrahari R, et al. SlNAC2 overexpression in Arabidopsis results in enhanced abiotic stress tolerance with alteration in glutathione metabolism. Protoplasma. 2019; 256: 1065-77.

[76] Wu C, Hao W, Yan L, et al. Postharvest melatonin treatment enhanced antioxidant activity and promoted GABA biosynthesis in yellow-flesh peach. Food Chem. 2023; 419: 136088.

[77] Arnao MB, Hernández-Ruiz J . Melatonin against environmental plant stressors: a review. Curr Protein Pept Sci. 2021; 22: 413-29.

[78] Sharma A, Wang J, Xu D, et al. Melatonin regulates the functional components of photosynthesis, antioxidant system, gene expression, and metabolic pathways to induce drought resistance in grafted Carya cathayensis plants. Sci Total Environ. 2020; 713: 136675.

[79] Sade N, Umnajkitikorn K, Rubio Wilhelmi MDM , et al. Delaying chloroplast turnover increases water-deficit stress tolerance through the enhancement of nitrogen assimilation in rice. J Exp Bot. 2018; 69: 867-78.

[80] Umnajkitikorn K, Sade N, Rubio Wilhelmi MDM, et al. Silencing of OsCV (chloroplast vesiculation) maintained photorespiration and N assimilation in rice plants grown under elevated CO2. Plant Cell Environ. 2020; 43: 920-33.

[81] Sakuma Y, Liu Q, Dubouzet JG, et al. DNA-binding specificity of the ERF/AP2 domain of Arabidopsis DREBs, transcription factors involved in dehydration- and cold-inducible gene expression. Biochem Biophys Res Commun. 2002; 290: 998-1009.

[82] Zhang L, Chen L, Pang S, et al. Function analysis of the ERF and DREB subfamilies in tomato fruit development and ripening. Front Plant Sci. 2022; 13: 849048.

[83] Chen J, Xia X, Yin W . A poplar DRE-binding protein gene, PeDREB2L, is involved in regulation of defense response against abiotic stress. Gene. 2011; 483: 36-42.

[84] Jiang L, Wang Y, Zhang S, et al. Tomato SlDREB1 gene conferred the transcriptional activation of drought-induced gene and an enhanced tolerance of the transgenic Arabidopsis to drought stress. Plant Growth Regul. 2017; 81: 131-45.

[85] Yu M, Liu J, Du B, et al. NAC transcription factor PwNAC11 activates ERD1 by interaction with ABF3 and DREB2A to enhance drought tolerance in transgenic Arabidopsis. Int J Mol Sci. 2021; 22: 6952.

[86] Yan H, Fu D, Zhu B, et al. Sprout vacuum-infiltration: a simple and efficient agroinoculation method for virus-induced gene silencing in diverse solanaceous species. Plant Cell Rep. 2012; 31: 1713-22.

[87] Luo S, Liu J, Shi K, et al. Integrated transcriptomic and metabolomic analyses reveal that MsSPHK1-a sphingosine kinase gene negatively regulates drought tolerance in alfalfa (Medicago sativa L.). Plant Physiol Biochem. 2025; 218: 109302.

[88] Zhu H, Wang J, Jiang D, et al. The miR157-SPL-CNR module acts upstream of bHLH101 to negatively regulate iron deficiency responses in tomato. J Integr Plant Biol. 2022; 64: 1059-75.

[89] Lai T, Wang X, Ye B, et al. Molecular and functional characterization of the SBP-box transcription factor SPL-CNR in tomato fruit ripening and cell death. J Exp Bot. 2020; 71: 2995-3011.

[90] Zhang X, Sun Y, Wu H, et al. Tobacco transcription factor NtWRKY70b facilitates leaf senescence via inducing ROS accumulation and impairing hydrogen sulfide biosynthesis. Int J Mol Sci. 2024; 25: 3686.

[91] Alexieva V, Sergiev I, Mapelli S, et al. The effect of drought and ultraviolet radiation on growth and stress markers in pea and wheat. Plant Cell Environ. 2001; 24: 1337-44.

[92] Hou L, Wang Z, Gong G, et al. Hydrogen sulfide alleviates manganese stress in Arabidopsis. Int J Mol Sci. 2022; 23: 5046.

[93] Park S, Byeon Y, Kim Y-S, et al. Kinetic analysis of purified recombinant rice N-acetylserotonin methyltransferase and peak melatonin production in etiolated rice shoots. J Pineal Res. 2013; 54: 139-44.