Characterization of the RBOH Gene Family and Analysis of Its Salt Stress Responses in Salvia miltiorrhiza

doi:10.13560/j.cnki.biotech.bull.1985.2026-0593

Abstract

Abstract:

Objective Respiratory burst oxidase homologs (RBOHs) are key rate-limiting enzymes that generate reactive oxygen species (ROS) in plants and play critical roles in stress responses. This study aims to investigate the regulatory characteristics of SmRBOHs under salt stress and their effects on active ingredient accumulation, thereby providing a theoretical basis for further functional analysis of SmRBOHs and for elucidating the molecular mechanisms underlying active component accumulation in Salvia miltiorrhiza. Method Using Arabidopsis thaliana RBOH protein sequences as a reference, we systematically identified members of the SmRBOH gene family, comprehensively analyzed their sequence characteristics, evolutionary relationships, and promoter cis-elements and transcription factor binding sites, and constructed a protein-protein interaction network. Transcriptome data were then used to screen for SmRBOHs co-expressed with genes involved in the biosynthetic pathways of salvianolic acids and tanshinones. Under salt stress treatments, real-time quantitative PCR (RT-qPCR) and other techniques were applied to analyze the correlations between SmRBOH expression levels, ROS content, and the accumulation of salvianolic acids and tanshinones. Result A total of six SmRBOHs were identified and classified into three subgroups based on phylogenetic analysis. Their promoter regions contained abundant cis-elements associated with abiotic stresses such as salt stress and abscisic acid (ABA), as well as transcription factor binding sites. The SmRBOHs exhibited tissue-specific expression patterns and differential responses to ABA. Additionally, these genes were co-expressed with key genes involved in the biosynthetic pathways of salvianolic acids and tanshinones. Under salt stress, the SmRBOH members displayed distinct expression patterns. Among them, SmRBOH2 expression was negatively correlated with H₂O₂ and tanshinone contents, but positively correlated with salvianolic acid accumulation. The other members showed opposite trends. Conclusion SmRBOHs are widely involved in hormone-mediated physiological regulation and salt stress responses in S. miltiorrhiza, and their transcriptional expression is modulated by various transcription factors. S. miltiorrhiza may regulate the biosynthesis of salvianolic acids and tanshinones through the ABA-mediated SnRK2-RBOH signaling module, thereby participating in adaptive responses to salt stress.

Key words: Salvia miltiorrhiza, RBOH gene family, salt stress, expression pattern, salvianolic acids, tanshinones, H₂O₂, correlation analysis

LI He-qin, YAN Heng-yu, YUAN Yu-xin, DUAN Yan-yan, LI Cai-li, SHAN Cheng-gang, JIANG Xu-wen. Characterization of the RBOH Gene Family and Analysis of Its Salt Stress Responses in Salvia miltiorrhiza[J]. Biotechnology Bulletin, 2026, 42(10): 238-249.

Figures/Tables 7

Fig. 1 Conserved domain (A), gene motif (B), and gene structure (C) of SmRBOHs

Fig. 2 Phylogenetic relationships of RBOH proteins from Salvia miltiorrhiza, Arabidopsis thaliana, Oryza sativa, Solanum lycopersicum, Scutellaria baicalensis, and Salvia divinorum

Fig. 3 Analysis of cis-acting elements, transcription factor binding sites, and protein-protein interactions of the SmRBOHsA: Types of cis-acting elements. B: Numbers of cis-acting elements. C: Transcription factor binding sites. D: Protein-protein interaction analysis

Fig. 4 Expression patterns of SmRBOHsA: Expression of SmRBOHs in different tissues. B: Co-expression pattern of SmRBOHs and genes involved in the biosynthesis pathway of tanshinone in response to ABA. C: Co-expression pattern of SmRBOHs and genes involved in the biosynthesis pathway of salvianolic acid in response to ABA

Fig. 5 Phenotypes of pot seedlings, root H2O2 content, and SmRBOHs expression of S. miltiorrhiza under salt stressA‒B: S. miltiorrhiza pot seedlings(A) and root H2O2 content (B) under salt stress. C‒H: Relative expression of SmRBOHs in roots of salt‑treated S. miltiorrhiza seedlings. *P<0.05. The same below

Fig. 6 Effect of salt stress on the contents of salvianolic acid (A) and tanshinone (B) in the roots of S. miltiorrhiza

Fig. 7 Correlation analysis of SmRBOHs expression in roots of S. miltiorrhiza under different treatments with H2O2, salvianolic acid, and tanshinone contentsCA: Caffeic acid. RA: Rosmarinic acid. SaA: Salvianolic acid A. SaB: Salvianolic acid B. DT-I: Dihydrotanshinone I. CT: Cryptotanshinone. T-I: Tanshinone I. T-IIA: Tanshinone IIA. **P<0.01

References 26

[27]	Rosignoli S, Paiardini A. Boosting the full potential of PyMOL with structural biology plugins [J]. Biomolecules, 2022, 12(12): 1764.
[28]	Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2^-ΔΔCt method [J]. Methods, 2001, 25(4): 402-408.
[29]	Yang JW, Ma CL, Jia R, et al. Different responses of two maize cultivars to Spodoptera frugiperda (Lepidoptera: Noctuidae) larvae infestation provide insights into their differences in resistance [J]. Front Plant Sci, 2023, 14: 1065891.
[30]	Huang Q, Sun MH, Yuan TP, et al. The AP2/ERF transcription factor SmERF1L1 regulates the biosynthesis of tanshinones and phenolic acids in Salvia miltiorrhiza [J]. Food Chem, 2019, 274: 368-375.
[31]	Zhang HY, Wang X, Yan A, et al. Evolutionary analysis of respiratory burst oxidase homolog (RBOH) genes in plants and characterization of ZmRBOHs [J]. Int J Mol Sci, 2023, 24(4): 3858.
[32]	Wang YH, Liu ZS, Li L, et al. The characteristics and expression analysis of the tomato SlRBOH gene family under exogenous phytohormone treatments and abiotic stresses [J]. Int J Mol Sci, 2024, 25(11): 5780.
[33]	Du LH, Jiang Z, Zhou YD, et al. Genome-wide identification and expression analysis of respiratory burst oxidase homolog (RBOH) gene family in eggplant (Solanum melongena L.) under abiotic and biotic stress [J]. Genes, 2023, 14(9): 1665.
[34]	Liu MM, Zhang Y, Pan T, et al. Genome-wide analysis of respiratory burst oxidase homolog gene family in pea (Pisum sativum L.) [J]. Front Plant Sci, 2023, 14: 1321952.
[35]	Kaur G, Pati PK. Analysis of cis-acting regulatory elements of respiratory burst oxidase homolog (Rboh) gene families in Arabidopsis and rice provides clues for their diverse functions [J]. Comput Biol Chem, 2016, 62: 104-118.
[36]	Zhang T, Wu AQ, Yue YP, et al. uORFs: important cis-regulatory elements in plants [J]. Int J Mol Sci, 2020, 21(17): 6238.
[37]	Shi M, Hua Q, Kai GY. Comprehensive transcriptomic analysis in response to abscisic acid in Salvia miltiorrhiza [J]. Plant Cell Tissue Organ Cult PCTOC, 2021, 147(2): 389-404.
[38]	de Zelicourt A, Colcombet J, Hirt H. The role of MAPK modules and ABA during abiotic stress signaling [J]. Trends Plant Sci, 2016, 21(8): 677-685.
[39]	Manna M, Rengasamy B, Sinha AK. Revisiting the role of MAPK signalling pathway in plants and its manipulation for crop improvement [J]. Plant Cell Environ, 2023, 46(8): 2277-2295.
[40]	Jalmi SK, Sinha AK. ROS mediated MAPK signaling in abiotic and biotic stress- striking similarities and differences [J]. Front Plant Sci, 2015, 6: 769.
[41]	Acharya BR, Jeon BW, Zhang W, et al. Open Stomata 1 (OST1) is limiting in abscisic acid responses of Arabidopsis guard cells [J]. New Phytol, 2013, 200(4): 1049-1063.
[42]	Begum K, Das A, Ahmed R, et al. Genome-wide analysis of respiratory burst oxidase homolog (Rboh) genes in Aquilaria species and insight into ROS-mediated metabolites biosynthesis and resin deposition [J]. Front Plant Sci, 2023, 14: 1326080.
[43]	Wang J, Liu XJ, Kang YD, et al. Functional analysis and interaction networks of Rboh in poplar under abiotic stress [J]. Front Plant Sci, 2025, 16: 1553057.
[44]	Pan L, Cai LR, Lu Y, et al. Genome-wide identification and expression analysis Pvrboh genes reveal their potential roles in salt tolerance and calcium signaling [J]. Plant Cell Physiol, 2026, 67(1): 8-19.
[45]	Anderssen S, Naômé A, Jadot C, et al. AURTHO: Autoregulation of transcription factors as facilitator of cis-acting element discovery [J]. Biochim Biophys Acta Gene Regul Mech, 2022, 1865(5): 194847.
[46]	Li WR, Yu JC, Wang RH, et al. Overexpression of SmGRAS5 enhances tolerance to abiotic stresses in Salvia miltiorrhiza [J]. Plant Physiol Biochem, 2025, 221: 109669.
[47]	Lv BB, Feng QQ, Shao GG, et al. The transcription factor Dof32 coordinates salvianolic acid biosynthesis and drought tolerance in Salvia miltiorrhiza [J]. Plant Physiol, 2025, 198(2): kiaf221.
[48]	Zhao GM, Shi QM, Han Y, et al. The physiological and biochemical responses of a medicinal plant (Salvia miltiorrhiza L.) to stress caused by various concentrations of NaCl [J]. PLoS One, 2014, 9(2): e89624.
[49]	Bi Q, Yao H, Wang F, et al. Integrative analysis of the pharmaceutical active ingredient and transcriptome of the aerial parts of Glycyrrhiza uralensis under salt stress reveals liquiritin accumulation via ABA-mediated signaling [J]. Mol Genet Genomics, 2022, 297(2): 333-343.
[50]	Zheng FX, Xu ZT, Deng XJ, et al. Comprehensive identification of aquaporins under salt stress, and functional characterization of DoTIP1-1 in Dendrobium officinale [J]. Plant Physiol Biochem, 2026, 231: 110952.
[1]	Wang PT, Liu WC, Han C, et al. Reactive oxygen species: Multidimensional regulators of plant adaptation to abiotic stress and development [J]. J Integr Plant Biol, 2024, 66(3): 330-367.
[2]	Mittler R, Zandalinas SI, Fichman Y, et al. Reactive oxygen species signalling in plant stress responses [J]. Nat Rev Mol Cell Biol, 2022, 23(10): 663-679.
[3]	Shabbir R, Javed T, Sun SR, et al. Reactive oxygen species-(ROS) in plant defense: jack-of-all-trades [J]. Crit Rev Biotechnol, 2026, 46(1): 152-160.
[4]	Sun LR, Zhao ZJ, Hao FS. NADPH oxidases, essential players of hormone signalings in plant development and response to stresses [J]. Plant Signal Behav, 2019, 14(11): 1657343.
[5]	Su CY, Ming QL, Rahman K, et al. Salvia miltiorrhiza: Traditional medicinal uses, chemistry, and pharmacology [J]. Chin J Nat Med, 2015, 13(3): 163-182.
[6]	Yang L, Huang XL, Wang ZH, et al. Research progress on the pharmacological properties of active ingredients from Salvia miltiorrhiza: a review [J]. Phytomedicine, 2025, 148: 157272.
[7]	刘佳欣, 张会龙, 邹荣松, 等. 不同类型盐生植物适应盐胁迫的生理生长机制及Na⁺逆向转运研究进展 [J]. 生物技术通报, 2023, 39(1): 59-71.
	Liu JX, Zhang HL, Zou RS, et al. Research progress in Na⁺ antiport and physiological growth mechanisms of differernt halophytes adapted to salt stress [J]. Biotechnol Bull, 2023, 39(1): 59-71.
[8]	孙良丹. 盐碱地特色中药农业新理念思考与实践 [J]. 华北理工大学学报: 自然科学版, 2024, 46(1): 1-7.
	Sun LD. Innovation and practice of new concepts in traditional Chinese medicine agriculture in saline-alkali land [J]. J N China Univ Sci Technol Nat Sci Ed, 2024, 46(1): 1-7.
[9]	黄姚姚, 申俊龙, 李洁. 逆境出质量: 从种质与环境的相互作用探讨道地药材的质量形成机制 [J]. 中草药, 2025, 56(8): 2945-2953.
	Huang YY, Shen JL, Li J. Adversity breeds quality: Exploring quality formation mechanism of genuine medicinal materials from interaction between germplasm and environment [J]. Chin Tradit Herb Drugs, 2025, 56(8): 2945-2953.
[10]	Qi JX, Yang SQ, Salam A, et al. OsRbohI regulates rice growth and development via jasmonic acid signalling [J]. Plant Cell Physiol, 2023, 64(6): 686-699.
[11]	Wang HQ, Zhao XY, Xuan W, et al. Rice roots avoid asymmetric heavy metal and salinity stress via an RBOH-ROS-auxin signaling cascade [J]. Mol Plant, 2023, 16(10): 1678-1694.
[12]	Chen YT, Zhang R, Wang RJ, et al. Overexpression of OsRbohH enhances heat and drought tolerance through ROS homeostasis and ABA mediated pathways in rice (Oryza sativa L.) [J]. Plants, 2024, 13(17): 2494.
[13]	Huang Y, Cao HS, Yang L, et al. Tissue-specific respiratory burst oxidase homolog-dependent H₂O₂ signaling to the plasma membrane H⁺-ATPase confers potassium uptake and salinity tolerance in Cucurbitaceae [J]. J Exp Bot, 2019, 70(20): 5879-5893.
[14]	Di QH, Zhou MD, Li YS, et al. Respiratory burst oxidase homolog 5.1 regulates H3K4me3 deposition and transcription after cold priming in cucumber [J]. Plant Physiol, 2025, 197(2): kiae461.
[15]	Zhang H, Liu XY, Tang C, et al. A respiratory burst oxidase homolog protein in Pyrus regulates stigmatic ROS production to control pollen tube growth [J]. Plant Physiol, 2025, 198(4): kiaf336.
[16]	Yang XQ, Zhang F, Yang M, et al. The NADPH-oxidase LsRbohC1 plays a role in lettuce (Lactuca sativa) seed germination [J]. Plant Physiol Biochem, 2020, 154: 751-757.
[17]	Huang WT, Zhang YL, Zhou JL, et al. The respiratory burst oxidase homolog protein D (GhRbohD) positively regulates the cotton resistance to Verticillium dahliae [J]. Int J Mol Sci, 2021, 22(23): 13041.
[18]	Chai SY, Yang ZY, Deng XX, et al. ZnO quantum dots alleviate salt stress in Salvia miltiorrhiza by enhancing growth, scavenging reactive oxygen species, and modulating stress-responsive genes [J]. Environ Pollut, 2024, 344: 123363.
[19]	Li HQ, Zhang JX, Mi XT, et al. Melatonin priming enhances the seed germination and seedling performance of Salvia miltiorrhiza under salt stress [J]. Ind Crops Prod, 2026, 242: 122972.
[20]	韩金龙, 张锋, 黄亭亭, 等. 26种药用植物黄河三角洲地区引种适应性评价 [J]. 现代中药研究与实践, 2017, 31(4): 5-7, 16.
	Han JL, Zhang F, Huang TT, et al. Evaluation on the adaptability of the introduction of 26 species of medicinal plants in the Yellow River Delta [J]. Res Pract Chin Med, 2017, 31(4): 5-7, 16.
[21]	窦家聪, 邢晓飞, 杨久涛, 等. 不同覆膜材料对黄河三角洲盐碱地丹参生长的影响 [J]. 中国农技推广, 2025, 41(12): 87-89, 96.
	Dou JC, Xing XF, Yang JT, et al. Effects of different film mulching materials on the growth of Salvia miltiorrhiza in saline-alkali land of the Yellow River Delta [J]. China Agric Technol Ext, 2025, 41(12): 87-89, 96.
[22]	Chen CJ, Wu Y, Li JW, et al. TBtools-II: a “one for all, all for one” bioinformatics platform for biological big-data mining [J]. Mol Plant, 2023, 16(11): 1733-1742.
[23]	Zhou CH, Lin CC, Xing PY, et al. SmGDB: genome database of Salvia miltiorrhiza, an important TCM Plant [J]. Genes Genom, 2022, 44(6): 699-707.
[24]	张鑫苗, 伍国强, 魏明. MAPK在植物响应逆境胁迫中的作用 [J]. 草业学报, 2024, 33(1): 182-197.
	Zhang XM, Wu GQ, Wei M. The role of MAPK in plant response to abiotic stress [J]. Acta Prataculturae Sin, 2024, 33(1): 182-197.
[25]	周颖, 白艳红, 王钰, 等. 丹参SnRK2基因家族的鉴定与表达 [J]. 分子植物育种, 2022, 20(7): 2233-2243.
	Zhou Y, Bai YH, Wang Y, et al. Genome-wide idenfication and expression pattern analysis of SnRK2 in Salvia miltiorrhiza Bunge [J]. Mol Plant Breed, 2022, 20(7): 2233-2243.
[26]	Xie YF, Ding ML, Zhang B, et al. Genome-wide characterization and expression profiling of MAPK cascade genes in Salvia miltiorrhiza reveals the function of SmMAPK3 and SmMAPK1 in secondary metabolism [J]. BMC Genomics, 2020, 21(1): 630.