Identification of auxin response factor in the pan-genome and their expression pattern analysis in cucumber

Qingchi Zhang; Ke Wang; Baihua Yang; Xinyi Guo; Lili Zhao; Chunhua Chen; Lina Wang; Zhonghai Ren; Qingchi Zhang; Ke Wang; Baihua Yang; Xinyi Guo; Lili Zhao; Chunhua Chen; Lina Wang; Zhonghai Ren

doi:10.3934/molsci.2025019

AIMS Molecular Science

2025, Volume 12, Issue 4: 318-340. doi: 10.3934/molsci.2025019

Previous Article Next Article

Research article

Identification of auxin response factor in the pan-genome and their expression pattern analysis in cucumber

Shandong Key Laboratory of Fruit and Vegetable Germplasm Innovation and Utilization, Shandong Collaborative Innovation Center of Fruit & Vegetable Quality and Efficient Production, Key Laboratory of Biology and Genetic Improvement of Horticultural Crops in Huang-Huai Region, Ministry of Agriculture, College of Horticultural Science and Engineering, Shandong Agricultural University, Tai'an 271018, China

Received: 15 July 2025 Revised: 10 September 2025 Accepted: 10 September 2025 Published: 26 September 2025

Auxin response factors (ARFs) are important transcription factors that regulate the expression of early genes in response to auxin. Auxin is a crucial phytohormone that controls plant growth and development. Even though ARF genes have been identified in the cucumber (Cucumis sativus) genome version 3.0, the features of this gene family are unclear in the cucumber pan-genome and in the newly assembled genome v4.0. We identified a total of 18 CsARF genes, including two CsARF13s in Gy14. All ARF proteins, except ARF5 and 7, showed differences in protein length; all of them showed amino acid variation in the cucumber pan-genome, including 13 accessions. We also analyzed their organ-specific expression and their expression patterns in fruit development and in response to biotic stresses. Importantly, we found that the overexpression of CsARF12 significantly enhanced the resistance of cucumber cotyledons to gray mold infection. Therefore, this study lays a foundation for understanding the biological functions of CsARF genes and provides CsARF12 as a candidate gene to improve the cucumber resistance to gray mold.
- cucumber,
- pan-genome,
- ARF gene,
- biotic stress response,
- gray mold
Citation: Qingchi Zhang, Ke Wang, Baihua Yang, Xinyi Guo, Lili Zhao, Chunhua Chen, Lina Wang, Zhonghai Ren. Identification of auxin response factor in the pan-genome and their expression pattern analysis in cucumber[J]. AIMS Molecular Science, 2025, 12(4): 318-340. doi: 10.3934/molsci.2025019

Related Papers:

Abstract

Auxin response factors (ARFs) are important transcription factors that regulate the expression of early genes in response to auxin. Auxin is a crucial phytohormone that controls plant growth and development. Even though ARF genes have been identified in the cucumber (Cucumis sativus) genome version 3.0, the features of this gene family are unclear in the cucumber pan-genome and in the newly assembled genome v4.0. We identified a total of 18 CsARF genes, including two CsARF13s in Gy14. All ARF proteins, except ARF5 and 7, showed differences in protein length; all of them showed amino acid variation in the cucumber pan-genome, including 13 accessions. We also analyzed their organ-specific expression and their expression patterns in fruit development and in response to biotic stresses. Importantly, we found that the overexpression of CsARF12 significantly enhanced the resistance of cucumber cotyledons to gray mold infection. Therefore, this study lays a foundation for understanding the biological functions of CsARF genes and provides CsARF12 as a candidate gene to improve the cucumber resistance to gray mold.

Acknowledgments

The authors thank the anonymous reviewers for their valuable suggestions to help improve this article. This work was supported by fundings from the National Natural Science Foundation of China (32172605), the Natural Science Foundation of Shandong Province (ZR2024MC032).

Conflict of interest

The authors declare no competing financial interest.

References

[1]	Calderon-Villalobos LI, Tan X, Zheng N, et al. (2010) Auxin perception--structural insights. Cold Spring Harb Perspect Biol 2: a005546. https://doi.org/10.1101/cshperspect.a005546
[2]	Yu Z, Zhang F, Friml J, et al. (2022) Auxin signaling: Research advances over the past 30 years. J Integr Plant Biol 64: 371-392. https://doi.org/10.1111/jipb.13225
[3]	Li Y, Han S, Qi Y (2023) Advances in structure and function of auxin response factor in plants. J Integr Plant Biol 65: 617-632. https://doi.org/10.1111/jipb.13392
[4]	Ulmasov T, Hagen G, Guilfoyle TJ (1999) Dimerization and DNA binding of auxin response factors. Plant J 19: 309-319. https://doi.org/10.1046/j.1365-313x.1999.00538.x
[5]	Guilfoyle TJ, Hagen G (2007) Auxin response factors. Curr Opin Plant Biol 10: 453-460. https://doi.org/10.1016/j.pbi.2007.08.014
[6]	Szemenyei H, Hannon M, Long JA (2008) TOPLESS mediates auxin-dependent transcriptional repression during Arabidopsis embryogenesis. Science 319: 1384-1386. https://doi.org/10.1126/science.1151461
[7]	Boer DR, Freire-Rios A, van den Berg WA, et al. (2014) Structural basis for DNA binding specificity by the auxin-dependent ARF transcription factors. Cell 156: 577-589. https://doi.org/10.1016/j.cell.2013.12.027
[8]	Franco-Zorrilla JM, López-Vidriero I, Carrasco JL, et al. (2014) DNA-binding specificities of plant transcription factors and their potential to define target genes. Proc Natl Acad Sci U S A 111: 2367-2372. https://doi.org/10.1073/pnas.1316278111
[9]	Wang S, Bai Y, Shen C, et al. (2010) Auxin-related gene families in abiotic stress response in Sorghum bicolor. Funct integr genomics 10: 533-546. https://doi.org/10.1007/s10142-010-0174-3
[10]	Zouine M, Fu Y, Chateigner-Boutin AL, et al. (2014) Characterization of the tomato ARF gene family uncovers a multi-levels post-transcriptional regulation including alternative splicing. PLoS One 9: e84203. https://doi.org/10.1371/journal.pone.0084203
[11]	Shen C, Wang S, Bai Y, et al. (2010) Functional analysis of the structural domain of ARF proteins in rice (Oryza sativa L.). J Exp Bot 61: 3971-3981. https://doi.org/10.1093/jxb/erq208
[12]	Okushima Y, Overvoorde PJ, Arima K, et al. (2005) Functional genomic analysis of the AUXIN RESPONSE FACTOR gene family members in Arabidopsis thaliana: Unique and overlapping functions of ARF7 and ARF19. Plant cell 17: 444-463. https://doi.org/10.1105/tpc.104.028316
[13]	Wilmoth JC, Wang S, Tiwari SB, et al. (2005) NPH4/ARF7 and ARF19 promote leaf expansion and auxin-induced lateral root formation. Plant J 43: 118-130. https://doi.org/10.1111/j.1365-313X.2005.02432.x
[14]	Tian C, Muto H, Higuchi K, et al. (2004) Disruption and overexpression of auxin response factor 8 gene of Arabidopsis affect hypocotyl elongation and root growth habit, indicating its possible involvement in auxin homeostasis in light condition. Plant J 40: 333-343. https://doi.org/10.1111/j.1365-313X.2004.02220.x
[15]	Wang S, Tiwari SB, Hagen G, et al. (2005) Auxin Response Factor₇ restores the expression of auxin-responsive genes in mutant Arabidopsis leaf mesophyll protoplasts. Plant Cell 17: 1979-1993. https://doi.org/10.1105/tpc.105.031096
[16]	Li J, Wu F, He Y, et al. (2022) Maize transcription factor ZmARF4 confers phosphorus tolerance by promoting root morphological development. Int J Mol Sci 23: 2361. https://doi.org/10.3390/ijms23042361
[17]	Gutierrez L, Bussell JD, Pacurar DI, et al. (2009) Phenotypic plasticity of adventitious rooting in Arabidopsis is controlled by complex regulation of Auxin Response Factor transcripts and microRNA abundance. Plant Cell 21: 3119-3132. https://doi.org/10.1105/tpc.108.064758
[18]	Sims K, Abedi-Samakush F, Szulc N, et al. (2021) OsARF11 promotes growth, meristem, seed, and vein formation during rice plant development. Int J Mol Sci 22: 4089. https://doi.org/10.3390/ijms22084089
[19]	Qi Y, Wang S, Shen C, et al. (2012) OsARF12, a transcription activator on auxin response gene, regulates root elongation and affects iron accumulation in rice (Oryza sativa). New Phytol 193: 109-120. https://doi.org/10.1111/j.1469-8137.2011.03910.x
[20]	Shen C, Wang S, Zhang S, et al. (2013) OsARF16, a transcription factor, is required for auxin and phosphate starvation response in rice (Oryza sativa L.). Plant Cell Environ 36: 607-620. https://doi.org/10.1111/pce.12001
[21]	Wang J, Wang R, Mao X, et al. (2019) TaARF4 genes are linked to root growth and plant height in wheat. Ann Bot 124: 903-915. https://doi.org/10.1093/aob/mcy218
[22]	Qiao L, Zhang W, Li X, et al. (2018) Characterization and expression patterns of auxin response factors in wheat. Front Plant Sci 9: 1395. https://doi.org/10.3389/fpls.2018.01395
[23]	Ellis CM, Nagpal P, Young JC, et al. (2005) Auxin Response Factor₁ and Auxin Response Factor₂ regulate senescence and floral organ abscission in Arabidopsis thaliana. Development 132: 4563-4574. https://doi.org/10.1242/dev.02012
[24]	Guan C, Wu B, Yu T, et al. (2017) Spatial auxin signaling controls leaf flattening in arabidopsis. Curr Biol 27: 2940-2950.E4. https://doi.org/10.1016/j.cub.2017.08.042
[25]	Harper RM, Stowe-Evans EL, Luesse DR, et al. (2000) The NPH4 locus encodes the auxin response factor ARF7, a conditional regulator of differential growth in aerial Arabidopsis tissue. Plant Cell 12: 757-770. https://doi.org/10.1105/tpc.12.5.757
[26]	Alexander L, Grierson D (2002) Ethylene biosynthesis and action in tomato: A model for climacteric fruit ripening. J Exp Bot 53: 2039-2055. https://doi.org/10.1093/jxb/erf072
[27]	Wu L, Tian Z, Zhang J (2018) Functional dissection of auxin response factors in regulating tomato leaf shape development. Front Plant Sci 9: 957. https://doi.org/10.3389/fpls.2018.00957
[28]	Uzair M, Long H, Zafar SA, et al. (2021) Narrow Leaf21, encoding ribosomal protein RPS3A, controls leaf development in rice. Plant Physiol 186: 497-518. https://doi.org/10.1093/plphys/kiab075
[29]	Liu S, Zhang Y, Feng Q, et al. (2018) Tomato Auxin Response Factor₅ regulates fruit set and development via the mediation of auxin and gibberellin signaling. Sci Rep 8: 2971. https://doi.org/10.1038/s41598-018-21315-y
[30]	Przemeck GK, Mattsson J, Hardtke CS, et al. (1996) Studies on the role of the Arabidopsis gene MONOPTEROS in vascular development and plant cell axialization. Planta 200: 229-237. https://doi.org/10.1007/bf00208313
[31]	Nagpal P, Ellis CM, Weber H, et al. (2005) Auxin response factors ARF6 and ARF8 promote jasmonic acid production and flower maturation. Development 132: 4107-4118. https://doi.org/10.1242/dev.01955
[32]	Yang J, Tian L, Sun MX, et al. (2013) Auxin Response Factor₁₇ is essential for pollen wall pattern formation in Arabidopsis. Plant Physiol 162: 720-731. https://doi.org/10.1104/pp.113.214940
[33]	Kumar R, Tyagi AK, Sharma AK (2011) Genome-wide analysis of auxin response factor (ARF) gene family from tomato and analysis of their role in flower and fruit development. Mol Genet Genomics 285: 245-260. https://doi.org/10.1007/s00438-011-0602-7
[34]	Hao Y, Hu G, Breitel D, et al. (2015) Auxin Response Factor SlARF2 is an essential component of the regulatory mechanism controlling fruit ripening in tomato. PLoS Genet 11: e1005649. https://doi.org/10.1371/journal.pgen.1005649
[35]	Sagar M, Chervin C, Mila I, et al. (2013) SlARF4, an auxin response factor involved in the control of sugar metabolism during tomato fruit development. Plant Physiol 161: 1362-1374. https://doi.org/10.1104/pp.113.213843
[36]	de Jong M, Wolters-Arts M, Schimmel BC, et al. (2015) Solanum lycopersicum Auxin Response Factor₉ regulates cell division activity during early tomato fruit development. J Exp Bot 66: 3405-3416. https://doi.org/10.1093/jxb/erv152
[37]	Liu X, Xu T, Dong X, et al. (2016) The role of gibberellins and auxin on the tomato cell layers in pericarp via the expression of ARFs regulated by miRNAs in fruit set. Acta Physiol Plant 38: 77. https://doi.org/10.1007/s11738-016-2091-0
[38]	Schruff MC, Spielman M, Tiwari S, et al. (2006) The Auxin Response Factor 2 gene of Arabidopsis links auxin signalling, cell division, and the size of seeds and other organs. Development 133: 251-261. https://doi.org/10.1242/dev.02194
[39]	Xing H, Pudake RN, Guo G, et al. (2011) Genome-wide identification and expression profiling of auxin response factor (ARF) gene family in maize. BMC Genomics 12: 178. https://doi.org/10.1186/1471-2164-12-178
[40]	Narise T, Kobayashi K, Baba S, et al. (2010) Involvement of auxin signaling mediated by IAA14 and ARF7/19 in membrane lipid remodeling during phosphate starvation. Plant Mol Biol 72: 533-544. https://doi.org/10.1007/s11103-009-9589-4
[41]	Wang S, Zhang S, Sun C, et al. (2014) Auxin response factor (OsARF12), a novel regulator for phosphate homeostasis in rice (Oryza sativa). New Phytol 201: 91-103. https://doi.org/10.1111/nph.12499
[42]	Shen C, Yue R, Yang Y, et al. (2014) OsARF16 is involved in cytokinin-mediated inhibition of phosphate transport and phosphate signaling in rice (Oryza sativa L.). PLoS One 9: e112906. https://doi.org/10.1371/journal.pone.0112906
[43]	Zhao S, Zhang ML, Ma TL, et al. (2016) Phosphorylation of ARF2 relieves its repression of transcription of the K⁺ transporter gene HAK5 in response to low potassium stress. Plant Cell 28: 3005-3019. https://doi.org/10.1105/tpc.16.00684
[44]	Sheng H, Cong DL, Ju HY (2020) Functional characterization of ZmHAK1 promoter and its regulatory transcription factors in maize. Mol Biol 54: 374-388. https://doi.org/10.31857/s0026898420030155
[45]	Xu D, Cao H, Fang W, et al. (2017) Linking hydrogen-enhanced rice aluminum tolerance with the reestablishment of GA/ABA balance and miRNA-modulated gene expression: A case study on germination. Ecotox Environ Safe 145: 303-312. https://doi.org/10.1016/j.ecoenv.2017.07.055
[46]	Liu X, Lin Y, Liu D, et al. (2017) MAPK-mediated auxin signal transduction pathways regulate the malic acid secretion under aluminum stress in wheat (Triticum aestivum L.). Sci Rep 7: 1620. https://doi.org/10.1038/s41598-017-01803-3
[47]	Jain M, Khurana JP (2009) Transcript profiling reveals diverse roles of auxin-responsive genes during reproductive development and abiotic stress in rice. FEBS J 276: 3148-3162. https://doi.org/10.1111/j.1742-4658.2009.07033.x
[48]	Zhou J, Wang X, Jiao Y, et al. (2007) Global genome expression analysis of rice in response to drought and high-salinity stresses in shoot, flag leaf, and panicle. Plant Mol Biol 63: 591-608. https://doi.org/10.1007/s11103-006-9111-1
[49]	Yuan J (2016) Studies on the function of Alkali stress responsive genes TaARF9 and TaNTL5 in wheat. Master Thesis, Shandong University, Jinan, China .
[50]	Qin Q, Li G, Jin L, et al. (2020) Auxin response factors (ARFs) differentially regulate rice antiviral immune response against rice dwarf virus. PLoS Pathog 16: e1009118. https://doi.org/10.1371/journal.ppat.1009118
[51]	Wang D, Pei K, Fu Y, et al. (2007) Genome-wide analysis of the auxin response factors (ARF) gene family in rice (Oryza sativa). Gene 394: 13-24. https://doi.org/10.1016/j.gene.2007.01.006
[52]	Wang Y, Deng D, Shi Y, et al. (2012) Diversification, phylogeny and evolution of auxin response factor (ARF) family: Insights gained from analyzing maize ARF genes. Mol Biol Rep 39: 2401-2415. https://doi.org/10.1007/s11033-011-0991-z
[53]	Li Y, Luo W, Sun Y, et al. (2022) Identification and expression analysis of miR160 and their target genes in cucumber. Biochem Genet 60: 127-152. https://doi.org/10.1007/s10528-021-10093-4
[54]	Guan J, Miao H, Zhang Z, et al. (2024) A near-complete cucumber reference genome assembly and Cucumber-DB, a multi-omics database. Mol Plant 2024, 17: 1178-1182. https://doi.org/10.1016/j.molp.2024.06.012
[55]	Li H, Wang S, Chai S, et al. (2022) Graph-based pan-genome reveals structural and sequence variations related to agronomic traits and domestication in cucumber. Nat Commun 13: 682. https://doi.org/10.1038/s41467-022-28362-0
[56]	Li Z, Zhang Z, Yan P, et al. (2011) RNA-Seq improves annotation of protein-coding genes in the cucumber genome. BMC Genomics 12: 540. https://doi.org/10.1186/1471-2164-12-540
[57]	Adhikari BN, Savory EA, Vaillancourt B, et al. (2012) Expression profiling of Cucumis sativus in response to infection by Pseudoperonospora cubensis. PLoS One 7: e34954. https://doi.org/10.1371/journal.pone.0034954
[58]	Kong W, Chen N, Liu T, et al. (2015) Large-scale transcriptome analysis of cucumber and Botrytis cinerea during infection. PLoS One 10: e0142221. https://doi.org/10.1371/journal.pone.0142221
[59]	Weigel D, Glazebrook J Transformation of Agrobacterium using the freeze-thaw method. Cold Spring Harb Protoc . https://doi.org/10.1101/pdb.prot4666
[60]	Liu SQ, Hu LF (2013) Genome-wide analysis of the auxin response factor gene family in cucumber. Genet Mol Res 12: 4317-4331. https://doi.org/10.4238/2013.April.2.1
[61]	Wang R, Li B, Zhang J, et al. (2024) Cytokinin oxidase gene CKX5 is modulated in the immunity of Arabidopsis to Botrytis cinerea. PLoS One 19: e0298260. https://doi.org/10.1371/journal.pone.0298260
[62]	Wang D, Xu H, Huang J, et al. (2020) The Arabidopsis CCCH protein C3H14 contributes to basal defense against Botrytis cinerea mainly through the WRKY33-dependent pathway. Plant Cell Environ 43: 1792-1806. https://doi.org/10.1111/pce.13771
[63]	Zhang M, Li W, Zhang T, et al. (2024) Botrytis cinerea-induced F-box protein 1 enhances disease resistance by inhibiting JAO/JOX-mediated jasmonic acid catabolism in Arabidopsis. Mol Plant 17: 297-311. https://doi.org/10.1016/j.molp.2023.12.020
[64]	Wang R, Liu K, Tang B, et al. (2023) The MADS-box protein SlTAGL1 regulates a ripening-associated SlDQD/SDH2 involved in flavonoid biosynthesis and resistance against Botrytis cinerea in post-harvest tomato fruit. Plant J 115: 1746-1757. https://doi.org/10.1111/tpj.16354
[65]	Wang J, Dai Y, Li X, et al. (2025) Tomato B-cell lymphoma2 (Bcl2)-associated athanogene 5 (SlBAG5) contributes negatively to immunity against necrotrophic fungus Botrytis cinerea through interacting with SlBAP1 and modulating catalase activity. Int J Biol Macromol 301: 140466. https://doi.org/10.1016/j.ijbiomac.2025.140466
[66]	Liu M, Zhang Q, Wang C, et al. (2020) CsWRKY10 mediates defence responses to Botrytis cinerea infection in Cucumis sativus. Plant Sci 300: 110640. https://doi.org/10.1016/j.plantsci.2020.110640

molsci-12-04-019-s001.pdf
molsci-12-04-019-s002.xlsx

Reader Comments

Your name:*

Email:*
© 2025 the Author(s), licensee AIMS Press. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0)