Genome-wide identification and cold-stress-responsive expression analysis of the NOX gene family in Cucumis melo
Received date: 2026-04-27
Revised date: 2026-06-11
Online published: 2026-07-28
Supported by
National College Students Innovation and Entrepreneurship Training Program(202513287028);Provincial College Students Innovation and Entrepreneurship Training Program(202513287026);Huzhou University College Students Innovation and Entrepreneurship Training Program(202513287025)
NADPH oxidases (NOXs) are crucial enzymes for reactive oxygen species (ROS) generation in plants and play vital roles in growth, development, and stress responses. To elucidate the sequence characteristics of the NOX gene family and its low-temperature response patterns in melon (Cucumis melo L.), this study conducted genome-wide identification and expression profiling of NOX family members using bioinformatics analysis, RNA-seq transcriptome sequencing, and real-time quantitative PCR (RT-qPCR). The results revealed that eight NOX members were identified in the melon genome, distributed across six chromosomes. All members harbored conserved domains including Ferric_reductase, FAD_binding_8, NAD_binding_6, and NADPH_Ox, and the encoded proteins were generally basic and hydrophilic. Phylogenetic analysis classified the NOX proteins into five subgroups. Synteny analysis indicated the presence of only one pair of intraspecific duplicated genes in melon, which was under purifying selection. The promoter regions contained multiple hormone- and stress-responsive cis-acting elements, with CmNOX2 and CmNOX4 harboring low-temperature responsive elements. Following treatment at 4℃ for 24 h and 48 h, leaf relative electrolyte leakage (REL) increased from 28.33% to 42.67% and 52.67%, respectively; transcriptome analysis identified 5,633 and 6,882 differentially expressed genes (DEGs), respectively. Cold-responsive genes exhibited significant differential expression, with SLAC1 and CPK19 showing sustained upregulation. RT-qPCR results demonstrated that the expression of CmNOX2, CmNOX5, CmNOX6, and CmNOX7 was significantly downregulated after low-temperature treatment, whereas CmNOX4 expression was significantly upregulated at 48 h. Integrating promoter elements and expression characteristics, CmNOX4 may represent an important candidate gene involved in melon low-temperature response. This study systematically characterized the structure, evolution, and expression patterns of the melon NOX gene family, identified candidate genes responsive to low temperature, and provides a reference for further investigation into the mechanisms underlying melon cold adaptation.
Xudong Qian , Yulu Zhong , Shuhui Zheng , Jianing Lü , Sushuang Liu . Genome-wide identification and cold-stress-responsive expression analysis of the NOX gene family in Cucumis melo[J]. Hereditas(Beijing), 2026 , 48(9) : 904 -920 . DOI: 10.16288/j.yczz.26-113
| [1] | Marino D, Dunand C, Puppo A, Pauly N. A burst of plant NADPH oxidases. Trends Plant Sci, 2012, 17(1): 9-15. |
| [2] | Chapman JM, Muhlemann JK, Gayomba SR, Muday GK. RBOH-dependent ROS synthesis and ROS scavenging by plant specialized metabolites to modulate plant development and stress responses. Chem Res Toxicol, 2019, 32(3): 370-396. |
| [3] | Kaya H, Takeda S, Kobayashi MJ, Kimura S, Iizuka A, Imai A, Hishinuma H, Kawarazaki T, Mori K, Yamamoto Y, Murakami Y, Nakauchi A, Abe M, Kuchitsu K. Comparative analysis of the reactive oxygen species- producing enzymatic activity of Arabidopsis NADPH oxidases. Plant J, 2019, 98(2): 291-300. |
| [4] | Chen QH, Yang GW. Signal function studies of ROS, especially RBOH-dependent ROS, in plant growth, development and environmental stress. J Plant Growth Regul, 2020, 39(1): 157-171. |
| [5] | Mittler R, Vanderauwera S, Gollery M, Van Breusegem F. Reactive oxygen gene network of plants. Trends Plant Sci, 2004, 9(10): 490-498. |
| [6] | Baxter A, Mittler R, Suzuki N. ROS as key players in plant stress signalling. J Exp Bot, 2014, 65(5): 1229-1240. |
| [7] | Chu-Puga Á, González-Gordo S, Rodríguez-Ruiz M, Palma JM, Corpas FJ. NADPH oxidase (Rboh) activity is up regulated during sweet pepper (Capsicum annuum L.) fruit ripening. Antioxidants (Basel), 2019, 8(1): 9. |
| [8] | Wang SS, Zhu XN, Lin JX, Zheng WJ, Zhang BT, Zhou JQ, Ni J, Pan ZC, Zhu SH, Ding WN. OsNOX3, encoding a NADPH oxidase, regulates root hair initiation and elongation in rice. Biol Plant, 2018, 62(4): 732-740. |
| [9] | Yang L, Ye CF, Zhao YT, Cheng XL, Wang YQ, Jiang YQ, Yang B. An oilseed rape WRKY-type transcription factor regulates ROS accumulation and leaf senescence in Nicotiana benthamiana and Arabidopsis through modulating transcription of RbohD and RbohF. Planta, 2018, 247(6): 1323-1338. |
| [10] | Khafi AS, Iranbakhsh A, Safipour Afshar A, Khavari Nejad RA. RBOH expression and ROS metabolism in Citrullus colocynthis under cadmium stress. Braz J Bot, 2020, 43(1): 35-43. |
| [11] | Piotrovskii MS, Shevyreva TA, Zhestkova IM, Trofimova MS. Activation of plasmalemmal NADPH oxidase in etiolated maize seedlings exposed to chilling temperatures. Russ J Plant Physiol, 2011, 58(2): 290-298. |
| [12] | Zhang YL, Zhang YW, Luo L, Lu CY, Kong WW, Cheng LB, Xu XY, Liu JH. Genome wide identification of respiratory burst oxidase homolog (Rboh) genes in Citrus sinensis and functional analysis of CsRbohD in cold tolerance. Int J Mol Sci, 2022, 23(2): 648. |
| [13] | Kabała K, Reda M, Wdowikowska A, Janicka M. Role of plasma membrane NADPH oxidase in response to salt stress in cucumber seedlings. Antioxidants (Basel), 2022, 11(8): 1534. |
| [14] | Chang YL, Li WY, Miao H, Yang SQ, Li R, Wang X, Li WQ, Chen KM. Comprehensive genomic analysis and expression profiling of the NOX gene families under abiotic stresses and hormones in plants. Genome Biol Evol, 2016, 8(3): 791-810. |
| [15] | Zhang ZB, Zhao YL, Feng XB, Luo ZY, Kong SW, Zhang C, Gong AD, Yuan HY, Cheng L, Wang XN. Genomic, molecular evolution, and expression analysis of NOX genes in soybean (Glycine max). Genomics, 2019, 111(4): 619-628. |
| [16] | Du LH, Jiang Z, Zhou YD, Shen L, He J, Xia X, Zhang LH, Yang X. Genome-wide identification and expression analysis of respiratory burst oxidase homolog (RBOH) gene family in eggplant (Solanum melongena L.) under abiotic and biotic stress. Genes (Basel), 2023, 14(9): 1665. |
| [17] | Di QH, Li YS, Li SZ, Shi AK, Zhou MD, Ren HZ, Yan Y, He CX, Wang J, Sun MT, Yu XC. Photosynthesis mediated by RBOH-dependent signaling is essential for cold stress memory. Antioxidants (Basel), 2022, 11(5): 969. |
| [18] | Wang GF, Li WQ, Li WY, Wu GL, Zhou CY, Chen KM. Characterization of rice NADPH oxidase genes and their expression under various environmental conditions. Int J Mol Sci, 2013, 14(5): 9440-9458. |
| [19] | Song ZQ, Chen C, Duan H, Yu T, Zhang YQ, Wei YN, Xu DC, Liu D. Identification of VcRBOH genes in blueberry and functional characterization of VcRBOHF in plant defense. BMC Genomics, 2025, 26(1): 153. |
| [20] | Hu CH, Wei XY, Yuan B, Yao LB, Ma TT, Zhang PP, Wang X, Wang PQ, Liu WT, Li WQ, Meng LS, Chen KM. Genome-wide identification and functional analysis of NADPH oxidase family genes in wheat during development and environmental stress responses. Front Plant Sci, 2018, 9: 906. |
| [21] | Cepauskas D, Miliute I, Staniene G, Gelvonauskiene D, Stanys V, Jesaitis AJ, Baniulis D. Characterization of apple NADPH oxidase genes and their expression associated with oxidative stress in shoot culture in vitro. Plant Cell Tiss Organ Cult, 2016, 124(3): 621-633. |
| [22] | Wang J, Liu XJ, Kang YD, Liu AZ, Li P. Functional analysis and interaction networks of Rboh in poplar under abiotic stress. Front Plant Sci, 2025, 16: 1553057. |
| [23] | Li DH, Wu D, Li SZ, Dai Y, Cao YP. Evolutionary and functional analysis of the plant-specific NADPH oxidase gene family in Brassica rapa L. R Soc Open Sci, 2019, 6(2): 181727. |
| [24] | Ye H, Liu HZ, Han M, Zhang NY, Feng XL, Gao T, Lei DF, Li HC, Gao ZM, Su Y, Chai M, Zhou HJ, Wang YL. The genome-wide identification, characterization, and expression profiles of the NADPH oxidase (NOX) gene family under drought and salt stress in Opisthopappus taihangensis (Asteraceae). Agronomy, 2024, 14(4): 653. |
| [25] | Yano R, Ariizumi T, Nonaka S, Kawazu Y, Zhong SL, Mueller L, Giovannoni JJ, Rose JKC, Ezura H. Comparative genomics of muskmelon reveals a potential role for retrotransposons in the modification of gene expression. Commun Biol, 2020, 3(1): 432. |
| [26] | Liu T, Shi JL, Li M, Ye XL, Qi HY. Trehalose triggers hydrogen peroxide and nitric oxide to participate in melon seedlings oxidative stress tolerance under cold stress. Environ Exp Bot, 2021, 184: 104379. |
| [27] | Korkmaz A, Dufault RJ. Developmental consequences of cold temperature stress at transplanting on seedling and field growth and yield. II. Muskmelon. J Am Soc Hortic Sci, 2001, 126(4): 410-413. |
| [28] | Korkmaz A, Dufault RJ. Influence of short-term cyclic cold temperature stress on muskmelon and honeydew yield. HortTechnology, 2003, 13(1): 67-70. |
| [29] | Nabwire S, Wakholi C, Faqeerzada MA, Arief MAA, Kim MS, Baek I, Cho BK. Estimation of cold stress, plant age, and number of leaves in watermelon plants using image analysis. Front Plant Sci, 2022, 13: 847225. |
| [30] | Yu JY, Wu S, Sun HH, Wang X, Tang XM, Guo SG, Zhang ZH, Huang SW, Xu Y, Weng YQ, Mazourek M, McGregor C, Renner SS, Branham S, Kousik C, Wechter WP, Levi A, Grumet R, Zheng Y, Fei ZJ. CuGenDBv2:an updated database for cucurbit genomics. Nucleic Acids Res, 2023, 51(D1): D1457-D1464. |
| [31] | Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCT method. Methods, 2001, 25(4): 402-408. |
| [32] | Sheng Y, Wang XY, Wang CY, Xu XY, Jiang LJ. Genome-wide identification and cold stress response analysis of the Rboh gene family in pomegranate (Punica granatum L.). Agric, 2025, 15(17): 1883. |
| [33] | Wang YH, Liu ZS, Li L, Pan XJ, Yao KD, Wei WY, Liao WB, Wang CL. The characteristics and expression analysis of the tomato SlRBOH gene family under exogenous phytohormone treatments and abiotic stresses. Int J Mol Sci, 2024, 25(11): 5780. |
| [34] | Zhang HY, Wang X, Yan A, Deng J, Xie YP, Liu SY, Liu DB, He L, Weng JF, Xu JY. Evolutionary analysis of respiratory burst oxidase homolog (RBOH) genes in plants and characterization of ZmRBOHs. Int J Mol Sci, 2023, 24(4): 3858. |
| [35] | Zhang JW, Xie YD, Ali B, Ahmed W, Tang Y, Li HX. Genome-wide identification, classification, evolutionary expansion and expression of Rboh family genes in pepper (Capsicum annuum L.). Trop Plant Biol, 2021, 14(3): 251-266. |
| [36] | Hu G, Liang DC. Bioinformatics of cassava NOX gene family and its expression analysis. J Southern Agric, 2019, 50(10): 2178-2187. |
| 胡广, 梁大成. 木薯NOX基因家族成员的生物信息学及其表达分析. 南方农业学报, 2019, 50(10): 2178-2187. | |
| [37] | Zhang YT, Li YL, He YW, Hu WJ, Zhang Y, Wang XR, Tang HR. Identification of NADPH oxidase family members associated with cold stress in strawberry. FEBS Open Bio, 2018, 8(4): 593-605. |
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