Auxin-induced AsARF16 complex orchestrates lncRNA125175-mediated ceRNA networks to regulate garlic somatic embryogenesis

Abstract

Somatic embryogenesis is a crucial biotechnological approach for effectively addressing garlic variety degeneration and improving yield and quality. Previous studies have demonstrated that the long noncoding RNA 125175 (lncRNA125175) is specifically induced and expressed during somatic embryogenesis, and may act as an endogenous target mimic of AsmiR393h to participate in the regulation of somatic embryogenesis. On this basis, the present study systematically elucidated the functions of the lncRNA125175/AsmiR393h/ AsTIR1 regulatory module and its upstream transcriptional mechanism. First, transient expression assays in tobacco leaves and protoplast experiments in garlic suggested that lncRNA125175 served as a competing endogenous RNA (ceRNA) to sequester AsmiR393h, thereby attenuating its post-transcriptional cleavage of the target gene AsTIR1. Promoter analysis revealed that all core components of this module contain auxin cis-acting elements, and the promoter activities of lncRNA125175 and AsTIR1 are significantly induced by exogenous auxin, suggesting that this ceRNA network is precisely regulated by auxin signaling. Further weighted gene co-expression network analysis identified the auxin response factor AsARF16 as a key upstream regulator. Yeast one-hybrid and two-hybrid assays indicated that AsARF16 can directly bind to the promoter of lncRNA125175, and interact with the transcription factor AsWRKY31 and the auxin signaling factor AsIAA33 to form a transcriptional activation complex. In conclusion, this study uncovers a cascade pathway from auxin signal perception (the AsARF16 complex) to transcriptional activation (lncRNA125175), followed by post-transcriptional ceRNA regulation. It systematically clarifies the molecular mechanism underlying its precise regulation of garlic somatic embryogenesis, providing a critical theoretical basis for the targeted improvement in garlic regeneration efficiency and genetic transformation systems.

Acknowledgements

This research was funded by National Natural Science Foundation of China (31372056; 31872125), Fundamental Research Funds for the Central Universities (YDZX2025038), Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD), Basic Research Funds for Central University-Science and Technology Poverty Alleviation Project (KJFP201702), and the Bioinformatics Center of Nanjing Agricultural University.

Author contributions

Y.B., Z.W., and R.Z. designed the study. Y.B., J.R., F.D., P.L., M.Z., and M.L. (Mengqian Li) performed the experiments. R.Z., F.J., and Z.W. supplied expertise and infrastructure for instrument use. Y.B., M.L. (Min Liu), H.W., and Y.C. analyzed data. Y.B., J.R., and R.Z. wrote the draft. All authors gave valuable comments and approved 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 declare there was no conflict of interest.

Supplementary material

Supplementary material is available at Horticulture Research online.

References

[1] Botas J, Fernandes N, Barros L. et al. A comparative study of black and white Allium sativum L.: nutritional composition and bioactive properties . Molecules. 2019; 24: 2194

[2] Namazi H . The role of garlic in the prevention of ischemia-reperfusion injury: a new mechanism. Mol Nutr Food Res. 2010; 52: 739

[3] Fan BL, He RF, Shang YT. et al. System construction of virus-free and rapid-propagation technology of baodi garlic (Allium sativum L.) . Sci Hortic. 2017; 225: 498-504

[4] Kereša S, Kurtović K, Ban SG. et al. Production of virus-free garlic plants through somatic embryogenesis. Agronomy. 2021; 11: 876

[5] Sivanesan I, Nayeem S, Venkidasamy B. et al. Genetic and epigenetic modes of the regulation of somatic embryogenesis: a review. Biologia Futur . 2022; 73: 259-77

[6] Castander-Olarieta A, Moncaleán P, Montalbán IA . Somatic embryogenesis in pines. Methods Mol Biol . 2022; 2527: 41-56

[7] Ikeuchi M, Favero DS, Sakamoto Y. et al. Molecular mechanisms of plant regeneration. Annu Rev Plant Biol. 2019; 70: 377-406

[8] Dharmasiri N, Dharmasiri S, Estelle M . The F-box protein TIR1 is an auxin receptor. Nature. 2005; 435: 441-5

[9] Karami O, De Jong H, Somovilla VJ. et al. Structure-activity relationship of 2,4-D correlates auxin activity with the induction of somatic embryogenesis in Arabidopsis thaliana . Plant J. 2023; 116: 1355-69

[10] Kepinski S, Leyser O . The Arabidopsis F-box protein TIR1 is an auxin receptor . Nature. 2005; 435: 446-51

[11] Friml J, Vieten A, Sauer M. et al. Efflux-dependent auxin gradients establish the apical-basal axis of Arabidopsis . Nature. 2003; 426: 147-53

[12] Friml J, Yang X, Michniewicz M. et al. A PINOID-dependent binary switch in apical-basal PIN polar targeting directs auxin efflux. Science. 2004; 306: 862-5

[13] Bartel DP . MicroRNAs: target recognition and regulatory functions. Cell. 2009; 136: 215-33

[14] Lin YL, Lai ZX . Comparative analysis reveals dynamic changes in miRNAs and their targets and expression during somatic embryogenesis in longan (Dimocarpus longan Lour.) . PLoS One. 2013; 8: e60337

[15] Sabana AA, Rajesh MK, Antony G . Dynamic changes in the expression pattern of miRNAs and associated target genes during coconut somatic embryogenesis. Planta. 2020; 251: 1-18

[16] Wu XM, Kou SJ, Liu YL. et al. Genome analysis of small RNAs in nonembryogenic and embryogenic tissues of citrus: microRNA-and siRNA-mediated transcript cleavage involved in somatic embryogenesis. Plant Biotechnol J. 2015; 13: 383-94

[17] Xu XQ, Zhang CY, Xu XQ. et al. Riboflavin mediates m ⁶A modification targeted by miR408, promoting early somatic embryogenesis in longan . Plant Physiol. 2023; 192: 1799-820

[18] Cordeiro D, Canhoto J, Correia S . Analysing the loss of embryogenic competence in long-term cell lines of Solanum betaceum Cav.: involvement of miR827, phosphate and sugar . BMC Plant Biol. 2025; 25: 851

[19] Jiang JJ, Zhu HT, Li N. et al. The miR393-target module regulates plant development and responses to biotic and abiotic stresses. Int J Mol Sci. 2022; 23: 9477

[20] Wójcik AM, Gaj MD . MiR393 contributes to the embryogenic transition induced in vitro in Arabidopsis via the modification of the tissue sensitivity to auxin treatment . Planta. 2016; 244: 231-43

[21] Yang XY, Wang LC, Yuan DJ. et al. Small RNA and degradome sequencing reveal complex miRNA regulation during cotton somatic embryogenesis. J Exp Bot. 2013; 64: 1521-36

[22] Huang JZ, Chen M, Chen D. et al. A peptide encoded by a putative lncRNA HOXB-AS3 suppresses colon cancer growth . Mol Cell. 2017; 68: 171-84

[23] Ebert MS, Neilson JR, Sharp PA . MicroRNA sponges: competitive inhibitors of small RNAs in mammalian cells. Nat Methods. 2007; 4: 721-6

[24] Fedak H, Palusinska M, Krzyczmonik K. et al. Control of seed dormancy in Arabidopsis by a cis-acting noncoding antisense transcript . Proc Natl Acad Sci U S A. 2016; 113: E7846-55

[25] Liu X, Li DY, Zhang DL. et al. A novel antisense long noncoding RNA, TWISTED LEAF, maintains LEAF blade flattening by regulating its associated sense R2R3-MYB gene in rice . New Phytol. 2018; 218: 774-88

[26] Qin T, Zhao HY, Cui P. et al. A nucleus-localized long non-coding RNA enhances drought and salt stress tolerance. Plant Physiol. 2017; 175: 1321-36

[27] Franco-Zorrilla JM, Valli A, Todesco M. et al. Target mimicry provides a new mechanism for regulation of microRNA activity. Nat Genet. 2007; 39: 1033-7

[28] Hong YH, Zhang YY, Cui J. et al. The lncRNA39896-miR166b- HDZs module affects tomato resistance to Phytophthora infestans . J Integr Plant Biol. 2022; 64: 13339

[29] Zhang Y, Hong Y, Liu Y. et al. Function identification of miR394 in tomato resistance to Phytophthora infestans . Plant Cell Rep. 2021; 40: 1831-44

[30] Zhang XP, Shen J, Xu QJ. et al. Long noncoding RNA lncRNA354 functions as a competing endogenous RNA of miR160b to regulate ARF genes in response to salt stress in upland cotton . Plant Cell Environ. 2021; 44: 3302-21

[31] Lin YL, Lin LX, Lai RL. et al. MicroRNA390-directed TAS3 cleavage leads to the production of tasiRNA-ARF3/4 during somatic embryogenesis in Dimocarpus longan Lour. Front Plant Sci. 2015; 6: 1119

[32] Sabana AA , Antony G , Gangaraj KP . et al. Regulation of coconut somatic embryogenesis: decoding the role of long non-coding RNAs. Plant Biotechnol Rep . 2024; 18: 33-44

[33] Gliwicka M, Nowak K, Balazadeh S. et al. Extensive modulation of the transcription factor transcriptome during somatic embryogenesis in Arabidopsis thaliana . PLoS One. 2013; 8: e69261

[34] Grunewald W, De Smet I, De Rybel B. et al. Tightly controlled WRKY23 expression mediates Arabidopsis embryo development . EMBO Rep. 2013; 14: 1136-42

[35] Chen XY, Zhang LY, Liu CB. et al. The transcription factor LpWRKY65 enhances embryogenic capacity through reactive oxygen species scavenging during somatic embryogenesis of larch. Plant Physiol. 2025; 198: kiaf286

[36] Li Y, Ma WH, Qi SZ. et al. Genome-wide characterization of the WRKY gene family and the role of LsfWRKY29 in regulating somatic embryogenesis in hybrid sweetgum (Liquidambar styraciflua x L. formosana) . Int J Biol Macromol. 2025; 293: 139287

[37] Xu XQ, Zhang CY, Tong N. et al. Transcriptome analysis revealed the regulatory mechanism of DIMBOA affecting early somatic embryogenesis in Dimocarpus longan Lour. Plants. 2025; 14: 442

[38] Yang Y, Wang N, Zhao SJ . Functional characterization of a WRKY family gene involved in somatic embryogenesis in Panax ginseng . Protoplasma. 2020; 257: 449-58

[39] Bai YH, Liu M, Zhou R. et al. Construction of ceRNA networks at different stages of somatic embryogenesis in garlic. Int J Mol Sci. 2023; 24: 5311

[40] Gao Y, Cui Y, Zhao RR. et al. Cryo-treatment enhances the embryogenicity of mature somatic embryos via the lncRNA-miRNA-mRNA network in white spruce. Int J Mol Sci. 2022; 23: 1111

[41] Rui Y, Song S, Li HY. et al. Functional analysis of the eTM-miR171- SCL6 module regulating somatic embryogenesis in Lilium pumilum DC. Fisch. Hortic Res. 2022; 9: 045

[42] Chen Y, Li X, Su LY. et al. Genome-wide identification and characterization of long non-coding RNAs involved in the early somatic embryogenesis in Dimocarpus longan Lour. BMC Genomics. 2018; 19: 805

[43] Du QG, Wang K, Zou C. et al. The PILNCR1-miR399 regulatory module is important for low phosphate tolerance in maize . Plant Physiol. 2018; 177: 1743-53

[44] Yang T, Ma HY, Zhang J. et al. Systematic identification of long noncoding RNAs expressed during light-induced anthocyanin accumulation in apple fruit. Plant J. 2019; 100: 572-90

[45] Chen YK, Xu XP, Liu ZX. et al. Global scale transcriptome analysis reveals differentially expressed genes involve in early somatic embryogenesis in Dimocarpus longan Lour. BMC Genomics. 2020; 21: 4

[46] Vondrakova Z, Dobrev PI, Pesek B. et al. Profiles of endogenous phytohormones over the course of Norway spruce somatic embryogenesis. Front Plant Sci. 2018; 9: 1283

[47] Guilfoyle TJ, Hagen G . Auxin response factors. Curr Opin Plant Biol . 2007; 10: 453-60

[48] Peiter E, Imani J, Yan F. et al. A novel procedure for gentle isolation and separation of intact infected and uninfected protoplasts from the central tissue of Vicia faba L. root nodules . Plant Cell Environ. 2003; 26: 1117-26