Transcription factor SlbHLH70 enhances drought tolerance in tomato

Abstract

Drought stress profoundly impacts plant productivity worldwide. The roles of basic helix–loop–helix (bHLH) transcription factors are critical in processes of plant growth, development, and stress management. However, roles of specific bHLH genes in tomato, particularly in relation to drought tolerance, remain poorly understood. This research identified SlbHLH70 as a factor that enhances drought tolerance in tomato. Transgenic lines overexpressing SlbHLH70 exhibited enhanced drought tolerance and improved post-stress recovery, whereas SlbHLH70 knockout mutants showed increased sensitivity to drought stress. Further study showed that SlbHLH70 directly regulated genes associated with abscisic acid (ABA) synthesis, ABA-mediated signal transduction, and root development. Our research identified SlbHLH70 as an important regulator of drought resistance in tomato, offering a valuable genetic target for enhancing crop resilience to water shortages.

Acknowledgements

This research was supported by Projects of Fund for Stable Support to Agricultural Sci-Tech Renovation (projects xjnkywdzc-2023001-06, xjnkywdzc-2025001-09, and xjnkywdzc-2025001-29), the earmarked fund for XJARS (project XJARS-07), and Special Project for Innovation Environment Construction in Xinjiang Uygur Autonomous Region-Construction of Science and Technology Innovation Bases (project PT2401).

Author contributions

Q.Y., J.W., and B.L. designed the work. A.L., Y.W., C.S., and M.M. participated in the experiment process and data analysis. A.L. wrote the manuscript. B.L., Y.L., Y.L., H.L., X.H., and B.W. provided guidance. B.L., M.Y., and Q.Y. revised the manuscript. All authors read and approved the final manuscript.

Data availability

The RNA-seq data supporting the findings of this study have been deposited in the CNGB Nucleotide Sequence Archive (CNSA) (https://www.cngb.org/), under project number CNP0009185.

Conflicts of interest statement

The authors declare that they have no conflicts of interest.

References

[1] Zhang H, Zhu J, Gong Z, et al. Abiotic stress responses in plants. Nat Rev Genet. 2022; 23: 104-19.

[2] Mccue KF, Hanson AD . Drought and salt tolerance: towards understanding and application. Trends Biotechnol. 1990; 8: 358-62.

[3] Liu Y, Xin X, Zheng J, et al. SlSAMS1 improves carbon and nitrogen metabolism in tomato under salt stress. Vegetable Research. 2025; 5: e021.

[4] Zhao H, Shin D, Zhu Y, et al. Bridging the knowledge gap: utilization of mediator subunits for crop improvement. Plant Cell Environ. 2025; 48: 213-25.

[5] Zhang J, Jia W, Yang J, et al. Role of ABA in integrating plant responses to drought and salt stresses. Field Crop Res. 2006; 97: 111-9.

[6] Feller A, Machemer K, Braun EL, et al. Evolutionary and comparative analysis of MYB and bHLH plant transcription factors. Plant J. 2011; 66: 94-116.

[7] Carretero-Paulet L, Galstyan A, Roig-Villanova I, et al. Genome-wide classification and evolutionary analysis of the bHLH family of transcription factors in Arabidopsis, poplar, rice, moss, and algae. Plant Physiol. 2010; 153: 1398-412.

[8] Blanc-Mathieu R, Dumas R, Turchi L, et al. Plant-TFClass: a structural classification for plant transcription factors. Trends Plant Sci. 2024; 29: 40-51.

[9] Pires N, Dolan L . Origin and diversification of basic-helix-loop-helix proteins in plants. Mol Biol Evol. 2010; 27: 862-74.

[10] Gao F, Dubos C . The Arabidopsis bHLH transcription factor family. Trends Plant Sci. 2023; 29: 668-80.

[11] Ding W, Yu Z, Tong Y, et al. A transcription factor with a bHLH domain regulates root hair development in rice. Cell Res. 2009; 19: 1309-11.

[12] Hatakeyama J, Kageyama R . Retinal Cell Fate Determination and bHLH Factors. In: Seminars in Cell & Developmental Biology. 2004, pp. 83-9.

[13] Guo J, Sun B, He H, et al. Current understanding of bHLH transcription factors in plant abiotic stress tolerance. Int J Mol Sci. 2021; 22: 4921.

[14] Lei P, Jiang Y, Zhao Y, et al. Functions of basic helix-loop-helix (bHLH) proteins in the regulation of plant responses to cold, drought, salt, and iron deficiency: a comprehensive review. J Agric Food Chem. 2024; 72: 10692-709.

[15] Liang B, Wan S, Ma Q, et al. A novel bHLH transcription factor PtrbHLH66 from trifoliate orange positively regulates plant drought tolerance by mediating root growth and ROS scavenging. Int J Mol Sci. 2022; 23: 15053.

[16] Song Y, Li S, Sui Y, et al. SbbHLH85, a bHLH member, modulates resilience to salt stress by regulating root hair growth in sorghum. Theor Appl Genet. 2022; 135: 201-16.

[17] Iuchi S, Kobayashi M, Taji T, et al. Regulation of drought tolerance by gene manipulation of 9-cis-epoxycarotenoid dioxygenase, a key enzyme in abscisic acid biosynthesis in Arabidopsis. Plant J. 2001; 27: 325-33.

[18] Schwartz SH, Qin X, Zeevaart JA . Elucidation of the indirect pathway of abscisic acid biosynthesis by mutants, genes, and enzymes. Plant Physiol. 2003; 131: 1591-601.

[19] Matilla AJ, Carrillo-Barral N, Rodríguez-Gacio MDC . An update on the role of NCED and CYP707a ABA metabolism genes in seed dormancy induction and the response to after-ripening and nitrate. J Plant Growth Regul. 2015; 34: 274-93.

[20] Quinet M, Angosto T, Yuste-Lisbona FJ, et al. Tomato fruit development and metabolism. Front Plant Sci. 2019; 10: 1554.

[21] Zhu Y, Zhu G, Xu R, et al. A natural promoter variation of SlBBX31 confers enhanced cold tolerance during tomato domestication. Plant Biotechnol J. 2023; 21: 1033-43.

[22] Wang Y, Wang H, Xin Y, et al. Genome-wide identification of SmJAZ gene family in eggplant and functional mechanism analysis of SmJAZ9 modulating high temperature and darkness stress-regulated anthocyanin biosynthesis. Vegetable Research. 2025; 5.

[23] Lin T, Zhu G, Zhang J, et al. Genomic analyses provide insights into the history of tomato breeding. Nat Genet. 2014; 46: 1220-6.

[24] Heim MA, Jakoby M, Werber M, et al. The basic helix-loop-helix transcription factor family in plants: a genome-wide study of protein structure and functional diversity. Mol Biol Evol. 2003; 20: 735-47.

[25] Zhang J, Huang Q, Zhong S, et al. Sperm cells are passive cargo of the pollen tube in plant fertilization. Nat Plants. 2017; 3: 1-5.

[26] Karas B, Amyot L, Johansen C, et al. Conservation of lotus and Arabidopsis basic helix-loop-helix proteins reveals new players in root hair development. Plant Physiol. 2009; 151: 1175-85.

[27] Bartlett A, O’Malley RC, Huang SC, et al. Mapping genome-wide transcription-factor binding sites using DAP-seq. Nat Protoc. 2017; 12: 1659-72.

[28] Ramírez F, Ryan DP, Grüning B, et al. DeepTools2: a next generation web server for deep-sequencing data analysis. Nucleic Acids Res. 2016; 44: W160.

[29] Sun Y, Han Y, Sheng K, et al. Single-cell transcriptomic analysis reveals the developmental trajectory and transcriptional regulatory networks of pigment glands in Gossypium bickii. Mol Plant. 2023; 16: 694-708.

[30] Zhao H, Yang M, Bishop J, et al. Identification and functional validation of super-enhancers in Arabidopsis thaliana. Proc Natl Acad Sci. 2022; 119: e2079639177.

[31] Zhao Y, Chan Z, Gao J, et al. ABA receptor PYL9 promotes drought resistance and leaf senescence. Proc Natl Acad Sci. 2016; 113: 1949-54.

[32] Liu L, Zhang J, Xu J, et al. SlMYC2 promotes SlLBD40-mediated cell expansion in tomato fruit development. Plant J. 2024; 118: 1872-88.

[33] Shahid A, White G, Diuwe J, et al. SLMAD: Statistical learning-based metric anomaly detection. In: International Conference on Service-Oriented Computing. Cham: Springer International Publishing, 2020, fanyu.

[34] Seo JS, Joo J, Kim MJ, et al. OsbHLH148, a basic helix-loop-helix protein, interacts with OsJAZ proteins in a jasmonate signaling pathway leading to drought tolerance in rice. Plant J. 2011; 65: 907-21.

[35] Kovak E, Blaustein-Rejto D, Qaim M . Genetically modified crops support climate change mitigation. Trends Plant Sci. 2022; 27: 627-9.

[36] Li Z, Liu C, Zhang Y, et al. The bHLH family member ZmPTF1 regulates drought tolerance in maize by promoting root development and abscisic acid synthesis. J Exp Bot. 2019; 70: 5471-86.

[37] Liu W, Tai H, Li S, et al. BHLH122 is important for drought and osmotic stress resistance in arabidopsis and in the repression of ABA catabolism. New Phytol. 2014; 201: 1192-204.

[38] Liang Y, Ma F, Li B, et al. A bHLH transcription factor, SlbHLH96, promotes drought tolerance in tomato. Hortic Res. 2022; 9: uhac198.

[39] Waseem M, Li Z . Overexpression of tomato SlbHLH22 transcription factor gene enhances fruit sensitivity to exogenous phytohormones and shortens fruit shelf-life. J Biotechnol. 2019; 299: 50-6.

[40] Hao Y, Zong X, Ren P, et al. Basic helix-loop-helix (bHLH) transcription factors regulate a wide range of functions in Arabidopsis. Int J Mol Sci. 2021; 22: 7152.

[41] Li J, Wang T, Han J, et al. Genome-wide identification and characterization of cucumber bHLH family genes and the functional characterization of CsbHLH041 in NaCl and ABA tolerance in Arabidopsis and cucumber. BMC Plant Biol. 2020; 20: 1-20.

[42] Pireyre M, Burow M . Regulation of MYB and bHLH transcription factors: a glance at the protein level. Mol Plant. 2015; 8: 378-88.

[43] Liu Y, Ji X, Nie X, et al. Arabidopsis AtbHLH112 regulates the expression of genes involved in abiotic stress tolerance by binding to their E-box and GCG-box motifs. New Phytol. 2015; 207: 692-709.

[44] Lian T, Xu Y, Li L, et al. Crystal structure of tetrameric Arabidopsis MYC2 reveals the mechanism of enhanced interaction with DNA. Cell Rep. 2017; 19: 1334-42.

[45] Zhu C, Lin Z, Liu Y, et al. A bamboo bHLH transcription factor PeRHL4 has dual functions in enhancing drought and phosphorus starvation tolerance. Plant Cell Environ. 2024; 47: 3015-29.

[46] Hu C, Wang M, Zhu C, et al. A transcriptional regulation of ERF15 contributes to ABA-mediated cold tolerance in tomato. Plant Cell Environ. 2024; 47: 1334- 47.

[47] Chong L, Xu R, Huang P, et al. The tomato OST1-VOZ1 module regulates drought-mediated flowering. Plant Cell. 2022; 34: 2001-18.

[48] de Ollas C, Dodd IC . Physiological impacts of ABA-JA interactions under water-limitation. Plant Mol Biol. 2016; 91: 641-50.

[49] Miyamoto K, Shimizu T, Mochizuki S, et al. Stress-induced expression of the transcription factor RERJ1 is tightly regulated in response to jasmonic acid accumulation in rice. Protoplasma. 2013; 250: 241-9.

[50] Fu J, Wu H, Ma S, et al. OsJAZ1 attenuates drought resistance by regulating JA and ABA signaling in rice. Front Plant Sci. 2017; 8: 2108.

[51] Rombauts S, Déhais P, Van Montagu M, et al. PlantCARE, a plant cis-acting regulatory element database. Nucleic Acids Res. 1999; 27: 295-6.

[52] Pan X, Welti R, Wang X . Quantitative analysis of major plant hormones in crude plant extracts by high-performance liquid chromatography-mass spectrometry. Nat Protoc. 2010; 5: 986-92.

[53] O’Malley RC, Huang SC, Song L, et al. Cistrome and epicistrome features shape the regulatory DNA landscape. Cell. 2016; 165: 1280-92.

[54] Li Q, Zhou L, Chen Y, et al. Phytochrome interacting factor regulates stomatal aperture by coordinating red light and abscisic acid. Plant Cell. 2022; 34: 4293-312.