Identification of specific miRNAs in early-stage mung bean (Vigna radiata) using DNA/AgNCs sensors and miRNAtome analysis

Abstract

MicroRNAs (miRNAs) are noncoding RNAs, ~21-24 nucleotides in length, that play a pivotal role in post-transcriptional gene regulation by inducing cleavage or translational repression of target mRNAs with complementary sequences. In this study, we identified miRNAs expressed during the early developmental stage of mung bean (Vigna radiata), a major legume crop, using small RNA sequencing (sRNA-seq), and analyzed their expression profiles across various mung bean tissues. Mung bean-specific miRNAs were found to be highly expressed in the aerial parts of seedlings, particularly in the leaves. Furthermore, the expression of these miRNAs was effectively validated using Tailed-Hoogsteen triplex DNA-encapsulated silver nanocluster (DNA/AgNC) sensors. The nanosensor enables rapid detection of target miRNAs within 30 min and is easy to apply for field-based assessments. The predicted target mRNAs of the identified miRNAs were associated with a range of biological processes relevant to early-stage development. This study highlights the potential of nanosensor-based approaches for the efficient identification of novel miRNAs in staple crops, offering a promising strategy to reduce the cost, time, and labor required during the transition from laboratory research to field applications.

Acknowledgements

This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF), funded by the Ministry of Science, ICT, and Future Planning NRF-RS-2023-NR077248 to S.W.Y. and supported in part by the NRF-RS-2022-NR074880 to H.J.J. This research was supported in part by the Brain Korea 21 (BK21) FOUR (Fostering Outstanding Universities for Research) program from the Ministry of Education (MOE, South Korea).

Authors contributions

H.J.J., Y.K.O., and S.W.Y. conceived the study and drafted the manuscript. Y.K.O., H.J.J., H.C.Y., M.Y.R., S.K.C., and M.K.C. performed molecular and biochemical analyses. Y.K.O., C.M., B.L., and M.Y.B. performed in silico analysis. J.H.K. contributed to the manuscript writing.

Data availability

The Illumina sRNA sequencing data will be deposited at ENA Accession PRJEB101758 Additional data supporting this article are available in the article and in its online supplementary material. Predicted miRNA candidates in mung bean are available at https://doi.org/10.6084/m9.figshare.29411522.

Conflicts of interest statement

No conflict of interest was declared.

Supplementary material

Supplementary material is available at Horticulture Research online.

References

[1] Huppertz M, Kachhap D, Dalai A. et al. Exploring the potential of mung bean: from domestication and traditional selection to modern genetic and genomic technologies in a changing world. J Agric Food Res. 2023; 14:100786

[2] Karim MA, Fukamachi H, Komori S. et al. Growth, yield and photosynthetic activity of Vigna radiata L. grown at different temperature and light levels. Plant Prod Sci. 2003; 6:43-9

[3] Singh KL, Mukherjee A, Kar RK. Early axis growth during seed germination is gravitropic and mediated by ros and calcium. J Plant Physiol. 2017; 216:181-7

[4] Somta P, Laosatit K, Yuan X. et al. Thirty years of mungbean genome research: where do we stand and what have we learned? Front Plant Sci. 2022; 13:944721

[5] Chen X. Small RNAs and their roles in plant development. Annu Rev Cell Dev Biol. 2009; 25:21-44

[6] Li S, Castillo-González C, Yu B. et al. The functions of plant small RNAs in development and in stress responses. Plant J. 2017; 90:654-70

[7] Zhang Z, Liu X, Guo X. et al. Arabidopsis ago3 predominantly recruits 24-nt small RNAs to regulate epigenetic silencing. Nat Plants. 2016; 2:1-7

[8] Zhu H, Hu F, Wang R. et al. Arabidopsis argonaute10 specif-ically sequesters mir166/165 to regulate shoot apical meris-tem development. Cell. 2011; 145:242-56

[9] Wang Z, Ma Z, Castillo-González C. et al. SWI2/SNF2 ATPase CHR2 remodels pri-miRNAs via serrate to impede miRNA pro-duction. Nature. 2018; 557:516-21

[10] Dong Q, Hu B, Zhang C. MicroRNAs and their roles in plant development. Front Plant Sci. 2022; 13:824240

[11] Jung HJ, Choi SW, Boo K-H. et al. Hyl1-cleavage subtilase 1 (HCS1) suppresses miRNA biogenesis in response to light-to-dark transition. Proc Natl Acad Sci. 2022; 119:e2116757119

[12] Park J, Giudicatti AJ, Bader ZE. et al. The high expres-sion of osmotically responsive gene15-histone deacetylase9 complex associates with hyponastic leaves 1 to modulate microRNA expression in response to abscisic acid signaling. Plant Cell. 2023; 35:2910-28

[13] Zhang F, Yang J, Zhang N. et al. Roles of microRNAs in abiotic stress response and characteristics regulation of plant. Front Plant Sci. 2022; 13:919243

[14] Zhang X, Yuan Y-R, Pei Y. et al. Cucumber mosaic virus-encoded 2b suppressor inhibits arabidopsis argonaute1 cleavage activity to counter plant defense. Genes Dev. 2006; 20:3255-68

[15] Qin Z, Li C, Mao L. et al. Novel insights from non-conserved microRNAs in plants. Front Plant Sci. 2014; 5:586

[16] Raza A, Charagh S, Karikari B. et al. MiRNAs for crop improve-ment. Plant Physiol Biochem. 2023; 201:107857

[17] Tang J, Chu C. MicroRNAs in crop improvement: fine-tuners for complex traits. Nat Plants. 2017; 3:1-11

[18] Xu Y, Chen X. MicroRNA biogenesis and stabilization in plants. Fundam Res. 2023; 3:707-17

[19] Oh YK, Yadavalli HC, Ryu MY. et al. Application of fluorescence i-motif DNA silver nanocluster sensor to visualize endoge-nous reactive oxygen species in plant cells. Plant Cell Rep. 2025; 44:1-15

[20] Yadavalli HC, Kim Y, Jung IL. et al. Energy transfer between i-motif DNA encapsulated silver nanoclusters and fluores-cein amidite efficiently visualizes the redox state of live cells. Small. 2024; 20:e2401629

[21] Shah P, Choi SW, Kim H-J. et al. Locking-to-unlocking sys-tem is an efficient strategy to design DNA/silver nanoclus-ters (AgNCs) probe for human miRNAs. Nucleic Acids Res. 2016; 44:e57-7

[22] Yadavalli HC, Park S, Kim Y. et al. Tailed-hoogsteen triplex DNA silver nanoclusters emit red fluorescence upon target miRNA sensing. Small. 2024; 20:e2306793

[23] Besnard F, Vernoux T, Hamant O. Organogenesis from stem cells in planta: multiple feedback loops integrating molecular and mechanical signals. Cell Mol Life Sci. 2011; 68:2885-906

[24] Zhou Y, Honda M, Zhu H. et al. Spatiotemporal sequestration of mir165/166 by arabidopsis argonaute10 promotes shoot apical meristem maintenance. Cell Rep. 2015; 10:1819-27

[25] Choi SW, Ryu MY, Viczian A. et al. Light triggers the miRNA-biogenetic inconsistency for de-etiolated seedling survivabil-ity in Arabidopsis thaliana. Mol Plant. 2020; 13:431-45

[26] Park SJ, Choi SW, Kim GM. et al. Light-stabilized FHA2 sup-presses miRNA biogenesis through interactions with DCL1 and HYL1. Mol Plant. 2021; 14:647-63

[27] Wang L YanX, LiY. et al. Prp4ka phosphorylates serrate for degradation via 20 s proteasome to fine-tune miRNA produc-tion in Arabidopsis. Sci Adv. 2022;8:eabm8435

[28] Zhu H, Zhou Y, Castillo-González C. et al. Bidirectional pro-cessing of pri-miRNAs with branched terminal loops by ara-bidopsis dicer-like1. Nat Struct Mol Biol. 2013; 20:1106-15

[29] Sherman BT, Hao M, Qiu J. et al. David: a web server for functional enrichment analysis and functional annotation of gene lists (2021 update). Nucleic Acids Res. 2022;50:W216-21

[30] Micheli F. Pectin methylesterases: cell wall enzymes with important roles in plant physiology. Trends Plant Sci. 2001; 6: 414-9

[31] Mano Y, Nemoto K, Suzuki M. et al. The AMI1 gene family: indole-3-acetamide hydrolase functions in auxin biosynthesis in plants. J Exp Bot. 2010; 61:25-32

[32] Zhang C, Ding Z, Wu K. et al. Suppression of jasmonic acid-mediated defense by viral-inducible microRNA319 facilitates virus infection in rice. Mol Plant. 2016; 9:1302-14

[33] Wang J-W, Wang L-J, Mao Y-B. et al. Control of root cap for-mation by microRNA-targeted auxin response factors in Ara-bidopsis. Plant Cell. 2005; 17:2204-16

[34] Koyama T, Sato F, Ohme-Takagi M. Roles of mir319 and TCP transcription factors in leaf development. Plant Physiol. 2017; 175:874-85

[35] Rodriguez RE, Mecchia MA, Debernardi JM. et al. Control of cell proliferation in Arabidopsis thaliana by microRNA mir396. Development. 2010; 137:103-12

[36] Yu S, Wang J-W. The crosstalk between microRNAs and gibberellin signaling in plants. Plant Cell Physiol. 2020; 61:1880-90

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

[38] Wilczynska A, Bushell M. The complexity of miRNA-mediated repression. Cell Death Differ. 2015; 22:22-33

[39] Kundu A, Paul S, Dey A. et al. High throughput sequencing reveals modulation of micrornas in Vigna mungo upon mung-bean yellow mosaic India virus inoculation highlighting stress regulation. Plant Sci. 2017; 257:96-105

[40] Abdelrahman M, Jogaiah S, Burritt DJ. et al. Legume genetic resources and transcriptome dynamics under abiotic stress conditions. Plant Cell Environ. 2018; 41:1972-83

[41] Buch DU, Sharma OA, Pable AA. et al. Characterization of microRNA genes from pigeonpea ( and understanding their involvement in drought stress. J Biotech-nol. 2020; 321:23-34

[42] Kumar S, Das M, Sadhukhan A. et al. Identification of dif-ferentially expressed mungbean miRNAs and their targets in response to drought stress by small RNA deep sequencing. Curr Plant Biol. 2022; 30:100246

[43] Paul S, Kundu A, Pal A. Identification and expression pro-filing of Vigna mungo microRNAs from leaf small RNA tran-scriptome by deep sequencing. J Integr Plant Biol. 2014; 56: 15-23

[44] Peláez P, Trejo MS, Iñiguez LP. et al. Identification and char-acterization of microRNAs in Phaseolus vulgaris by high-throughput sequencing. BMC Genomics. 2012; 13:1-18

[45] Shao Z-Q, Zhang Y-M, Hang Y-Y. et al. Long-term evolution of nucleotide-binding site-leucine-rich repeat genes: under-standing gained from and beyond the legume family. Plant Physiol. 2014; 166:217-34

[46] Han M-H, Goud S, Song L. et al. The arabidopsis double-stranded RNA-binding protein HYL1 plays a role in microRNA-mediated gene regulation. Proc Natl Acad Sci. 2004; 101:1093-8

[47] Friedländer MR, Chen W, Adamidi C. et al. Discovering microR-NAs from deep sequencing data using mirdeep. Nat Biotech-nol. 2008; 26:407-15

[48] Rehmsmeier M, Steffen P, Höchsmann M. et al. Fast and effec-tive prediction of microRNA/target duplexes. Rna. 2004; 10: 1507-17

[49] Huang DW, Sherman BT, Lempicki RA. Systematic and inte- grative analysis of large gene lists using David bioinformatics resources. Nat Protoc. 2009; 4:44-57

[50] Zhou Y, Liu H, Wu T. et al. Screening of reference genes under biotic stress and hormone treatment of mung bean (Vigna radiata) by quantitative real-time PCR. Genes. 2023; 14:1739