Abstract
Neem (Azadirachta indica) is a very popular traditional medicinal plant used since ancient times to treat numerous ailments. MicroRNAs (miRNAs) are highly conserved, non-coding, short RNA molecules that play important regulatory roles in plant development and metabolism. In this study, deploying a high stringent genome-wide computational-based approach and following a set of strict filtering norms a total of 44 potential conserved neem miRNAs belonging to 21 families and their corresponding 48 potential target transcripts were identified. Important targets include Squamosa promoter binding protein-like proteins, NAC, Scarecrow proteins, Auxin response factor, and F-box proteins. A biological network has also been developed to understand the miRNA-mediated gene regulation using the minimum free energy (MFE) values of the miRNA-target interaction. Moreover, six selected miRNAs were reported to be involved in secondary metabolism in other plant species (miR156a, miR156l, miR160, miR164, miR171, miR395) were validated by qPCR and their tissue-specific differential expression pattern was observed in leaves and stem. Except for ain-miR395, all the other miRNAs were found overexpressed in the stem as compared to leaves. To the best of our knowledge, this is the first report of neem miRNAs and we believe the finding of the present study will be useful for the functional genomic study of medicinal plants.
This is a preview of subscription content, log in via an institution to check access.
References
Alptekin B, Akpinar BA, Budak H (2017) A comprehensive prescription for plant miRNA identification. Front Plant Sci 7:2058
Álvarez-Caballero JM, Coy-Barrera E (2019) Chemical and antifungal variability of several accessions of azadirachta indica A. Juss. from six locations across the colombian caribbean coast: identification of antifungal azadirone limonoids. Plants 8:555
Bartel DP (2004) MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 116:281–297. https://doi.org/10.1016/S0092-8674(04)00045-5
Bellato M, De Marchi D, Gualtieri C et al (2019) A bioinformatics approach to explore MicroRNAs as tools to bridge pathways between plants and animals is DNA damage response (DDR) a potential target process? Front Plant Sci 10:1535
Bhambhani S, Lakhwani D, Gupta P et al (2017) Transcriptome and metabolite analyses in Azadirachta indica: identification of genes involved in biosynthesis of bioactive triterpenoids. Sci Rep 7:5043. https://doi.org/10.1038/s41598-017-05291-3
Boke H, Ozhuner E, Turktas M et al (2015) Regulation of the alkaloid biosynthesis by miRNA in opium poppy. Plant Biotechnol J 13:409–420. https://doi.org/10.1111/pbi.12346
Bonnet E, Wuyts J, Rouzé P, Van de Peer Y (2004) Evidence that microRNA precursors, unlike other non-coding RNAs, have lower folding free energies than random sequences. Bioinformatics 20:2911–2917. https://doi.org/10.1093/bioinformatics/bth374
Budak H, Akpinar BA (2015) Plant miRNAs: biogenesis, organization and origins. Funct Integr Genomics 15:523–531
Cesa S, Sisto F, Zengin G et al (2019) Phytochemical analyses and pharmacological screening of neem oil. South Afr J Bot 120:331–337. https://doi.org/10.1016/j.sajb.2018.10.019
Chen Q, Yan J, Meng X et al (2017) Molecular cloning, characterization, and functional analysis of acetyl-CoA C-acetyltransferase and mevalonate kinase genes involved in terpene trilactone biosynthesis from ginkgo biloba. Molecules 22:74
Dai X, Zhuang Z, Zhao PX (2011) Computational analysis of miRNA targets in plants: current status and challenges. Brief Bioinform 12:115–121
Dai X, Zhuang Z, Zhao PX (2018) psRNATarget: a plant small RNA target analysis server (2017 release). Nucleic Acids Res 46:W49–W54
Darzentas N (2010) Circoletto visualizing sequence similarity with circos. Bioinformatics 26:2620–2621
Dezulian T, Palatnik JF, Huson D, Weigel D (2005) Conservation and divergence of microRNA families in plants. Genome Biol 6:1–25
Elgorashi EE, McGaw LJ (2019) African plants with in vitro anti-inflammatory activities: a review. South Afr J Bot 126:142–169. https://doi.org/10.1016/j.sajb.2019.06.034
Feder A, Burger J, Gao S et al (2015) A kelch domain-containing f-box coding gene negatively regulates flavonoid accumulation in muskmelon. Plant Physiol 169:1714–1726. https://doi.org/10.1104/pp.15.01008
Gupta OP, Karkute SG, Banerjee S et al (2017) Contemporary understanding of miRNA-based regulation of secondary metabolites biosynthesis in plants. Front Plant Sci 8:374
Ho C-L, Tan Y-C, Yeoh K-A et al (2016) De novo transcriptome analyses of host-fungal interactions in oil palm Elaeis guineensis Jacq. BMC Genom 17:66. https://doi.org/10.1186/s12864-016-2368-0
Jain A, Das S (2016) Synteny and comparative analysis of miRNA retention, conservation, and structure across Brassicaceae reveals lineage- and sub-genome-specific changes. Funct Integr Genomics 16:253–268. https://doi.org/10.1007/s10142-016-0484-1
Kozomara A, Birgaoanu M, Griffiths-Jones S (2019) miRBase: from microRNA sequences to function. Nucleic Acids Res 47:D155–D162
Krishnan NM, Pattnaik S, Jain P et al (2012) A draft of the genome and four transcriptomes of a medicinal and pesticidal angiosperm Azadirachta indica. BMC Genomics 13:464. https://doi.org/10.1186/1471-2164-13-464
Kundu A, Paul S, Dey A, Pal A (2017) High throughput sequencing reveals modulation of microRNAs in Vigna mungo upon Mungbean yellow mosaic India virus inoculation highlighting stress regulation. Plant Sci 257:96–105. https://doi.org/10.1016/j.plantsci.2017.01.016
Kuravadi NA, Yenagi V, Rangiah K et al (2015) Comprehensive analyses of genomes, transcriptomes and metabolites of neem tree. PeerJ 3:e1066
Lee W-K, Namasivayam P, Ong Abdullah J, Ho C-L (2017) Transcriptome profiling of sulfate deprivation responses in two agarophytes Gracilaria changii and Gracilaria salicornia (Rhodophyta). Sci Rep 7:46563. https://doi.org/10.1038/srep46563
Li X, Xin X, Li J, Cui Y, Hou Y, Zhai L, Wang X, Fu Y, Liu R, Bian S (2017) Conservation and diversification of the miR166 family in soybean and potential roles of newly identified miR166s. BMC Plant Biol 17:1–18
Li H, Lin Q, Yan M et al (2021) Relationship between secondary metabolism and miRNA for important flavor compounds in different tissues of tea plant (Camellia sinensis) As revealed by genome-wide miRNA analysis. J Agric Food Chem 69:2001–2012
Liu M-Y, Wu X-M, Long J-M, Guo W-W (2017) Genomic characterization of miR156 and Squamosa promoter binding protein-like genes in sweet orange (Citrus sinensis). Plant Cell Tissue Organ Cult 130:103–116
Mao W, Li Z, Xia X et al (2012) A combined approach of high-throughput sequencing and degradome analysis reveals tissue specific expression of micrornas and their targets in cucumber. PLoS ONE 7:e33040
Mica E, Piccolo V, Delledonne M et al (2009) High throughput approaches reveal splicing of primary microRNA transcripts and tissue specific expression of mature microRNAs in vitis vinifera. BMC Genomics 10:558. https://doi.org/10.1186/1471-2164-10-558
Morishita T, Kojima Y, Maruta T et al (2009) arabidopsis NAC transcription factor, ANAC078, regulates flavonoid biosynthesis under high-light. Plant Cell Physiol 50:2210–2222. https://doi.org/10.1093/pcp/pcp159
Naya L, Paul S, Valdés-López O et al (2014) Regulation of copper homeostasis and biotic interactions by microRNA 398b in common bean. PLoS ONE 9:e84416
Panda D, Dehury B, Sahu J et al (2014) Computational identification and characterization of conserved miRNAs and their target genes in garlic (Allium sativum L.) expressed sequence tags. Gene 537:333–342. https://doi.org/10.1016/j.gene.2014.01.010
Parry G, Estelle M (2006) Auxin receptors: a new role for F-box proteins. Curr Opin Cell Biol 18:152–156. https://doi.org/10.1016/j.ceb.2006.02.001
Paul S (2017) Identification and characterization of microRNAs and their targets in high-altitude stress-adaptive plant maca (Lepidium meyenii Walp). 3 Biotech 7:103. https://doi.org/10.1007/s13205-017-0734-5
Paul S, Kundu A, Pal A (2011) Identification and validation of conserved microRNAs along with their differential expression in roots of Vigna unguiculata grown under salt stress. Plant Cell Tissue Organ Cult 105:233–242. https://doi.org/10.1007/s11240-010-9857-7
Paul S, Kundu A, Pal A (2014) Identification and expression profiling of Vigna mungo microRNAs from leaf small RNA transcriptome by deep sequencing. J Integr Plant Biol 56:15–23. https://doi.org/10.1111/jipb.12115
Paul S, de la Fuente-Jiménez JL, Manriquez CG, Sharma A (2020) Identification, characterization and expression analysis of passion fruit (Passiflora edulis) microRNAs. 3 Biotech 10:25. https://doi.org/10.1007/s13205-019-2000-5
Pillet J, Yu H-W, Chambers AH et al (2015) Identification of candidate flavonoid pathway genes using transcriptome correlation network analysis in ripe strawberry (Fragaria × ananassa) fruits. J Exp Bot 66:4455–4467. https://doi.org/10.1093/jxb/erv205
Pirrò S, Zanella L, Kenzo M et al (2016) MicroRNA from Moringa oleifera: identification by high throughput sequencing and their potential contribution to plant medicinal value. PLoS ONE 11:e0149495
Ramachandran S, Hiratsuka K, Chua N-H (1994) Transcription factors in plant growth and development. Curr Opin Genet Dev 4:642–646. https://doi.org/10.1016/0959-437X(94)90129-Q
Roy S, Nath D, Paul P, Chakraborty S (2020) Computational identification of conserved microRNAs and functional annotation of their target genes in Citrus limon identification of microRNA in Citrus limon. South Afr J Bot 130:109–116. https://doi.org/10.1016/j.sajb.2019.12.009
Samad AFA, Kamaroddin MF, Sajad M (2020) Cross-kingdom regulation by plant microRNAs provides novel insight into gene regulation. Adv Nutr 12:197–211
Sarah R, Tabassum B, Idrees N, Hussain MK (2019) Bio-active compounds isolated from neem tree and their applications. Natural bio-active compounds. Springer, Berlin, pp 509–528
Sharma A, Bejerano PIA, Maldonado IC et al (2019) Genome-wide computational prediction and experimental validation of quinoa (Chenopodium quinoa) microRNAs. Can J Plant Sci 99:666–675. https://doi.org/10.1139/cjps-2018-0296
Shinde H, Dudhate A, Anand L et al (2020) Small RNA sequencing reveals the role of pearl millet miRNAs and their targets in salinity stress responses. South Afr J Bot 132:395–402. https://doi.org/10.1016/j.sajb.2020.06.011
Subapriya R, Nagini S (2005) Medicinal properties of neem leaves: a review. Curr Med Chem Agents 5:149–156
Sun Y, Qiu Y, Duan M et al (2017) Identification of anthocyanin biosynthesis related microRNAs in a distinctive Chinese radish (Raphanus sativus L.) by high-throughput sequencing. Mol Genet Genomics 292:215–229
Tiwari R, Verma AK, Chakraborty S et al (2014) Neem (Azadirachta indica) and its potential for safeguarding health of animals and humans: a review. J Biol Sci 14:110
Unver T, Budak H (2009) Conserved microRNAs and their targets in model grass species Brachypodium distachyon. Planta 230:659–669. https://doi.org/10.1007/s00425-009-0974-7
Varkonyi-Gasic E, Gould N, Sandanayaka M et al (2010) Characterisation of microRNAs from apple (Malus domestica’Royal Gala’) vascular tissue and phloem sap. BMC Plant Biol 10:159. https://doi.org/10.1186/1471-2229-10-159
Wu B, Wang M, Ma Y et al (2012) High-Throughput sequencing and characterization of the small RNA transcriptome reveal features of novel and conserved microRNAs in panax ginseng. PLoS ONE 7:e44385
Wu L, Liu S, Qi H et al (2020) Research progress on plant long non-coding RNA. Plants 9:408
Yang X, Li L (2012) Analyzing the microRNA transcriptome in plants using deep sequencing data. Biology Basel 1:297–310. https://doi.org/10.3390/biology1020297
Yu H, Cong L, Zhu Z et al (2015) Identification of differentially expressed microRNA in the stems and leaves during sugar accumulation in sweet sorghum. Gene 571:221–230. https://doi.org/10.1016/j.gene.2015.06.056
Zhang B, Pan X, Wang Q et al (2006b) Computational identification of microRNAs and their targets. Comput Biol Chem 30:395–407
Zhang B, Pan X, Cobb GP, Anderson TA (2006a) Plant microRNA: a small regulatory molecule with big impact. Dev Biol 289:3–16. https://doi.org/10.1016/j.ydbio.2005.10.036
Zhang S, Yan S, Zhao J et al (2019) Identification of miRNAs and their target genes in larix olgensis and verified of differential expression miRNAs. BMC Plant Biol 19:247. https://doi.org/10.1186/s12870-019-1853-4
Zhou L, Liu Y, Liu Z et al (2010) Genome-wide identification and analysis of drought-responsive microRNAs in Oryza sativa. J Exp Bot 61:4157–4168. https://doi.org/10.1093/jxb/erq237
Acknowledgements
The research facility provided by the Departamento Regional de Bioingenieria, Queretaro is thankfully acknowledged
Author information
Authors and Affiliations
Contributions
Conceptualization, experimental design, data analysis, writing, editing SP.; Experimental procedures, data collection and analysis, writing, PRP.; Bioinformatics and statistical analysis, critical writing, reviewing ASr, PI.-AB., SR, ASh; Project administration ASh. All authors have read and agreed to the published version of the manuscript.
Corresponding authors
Ethics declarations
Conflict of interest
The authors declare no conflict of interest.
Supplementary Information
Below is the link to the electronic supplementary material.
Rights and permissions
About this article
Cite this article
Paul, S., Reyes-Pérez, P., Angulo-Bejarano, P.I. et al. Characterization of microRNAs from neem (Azadirachta indica) and their tissue-specific expression study in leaves and stem. 3 Biotech 11, 277 (2021). https://doi.org/10.1007/s13205-021-02839-z
Received:
Accepted:
Published:
DOI: https://doi.org/10.1007/s13205-021-02839-z