{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,8]],"date-time":"2026-08-08T04:43:16Z","timestamp":1786164196644,"version":"build-2736575974"},"reference-count":45,"publisher":"MDPI AG","issue":"15","license":[{"start":{"date-parts":[[2026,8,6]],"date-time":"2026-08-06T00:00:00Z","timestamp":1785974400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100003453","name":"Natural Science Foundation of Guangdong Province","doi-asserted-by":"crossref","award":["2025A1515012337"],"award-info":[{"award-number":["2025A1515012337"]}],"id":[{"id":"10.13039\/501100003453","id-type":"DOI","asserted-by":"crossref"}]},{"DOI":"10.13039\/501100003453","name":"Natural Science Foundation of Guangdong Province","doi-asserted-by":"crossref","award":["2026A1515012922"],"award-info":[{"award-number":["2026A1515012922"]}],"id":[{"id":"10.13039\/501100003453","id-type":"DOI","asserted-by":"crossref"}]},{"DOI":"10.13039\/501100012245","name":"Guangdong Science and Technology Program","doi-asserted-by":"crossref","award":["2022DZXHT072"],"award-info":[{"award-number":["2022DZXHT072"]}],"id":[{"id":"10.13039\/501100012245","id-type":"DOI","asserted-by":"crossref"}]},{"DOI":"10.13039\/501100012245","name":"Guangdong Science and Technology Program","doi-asserted-by":"crossref","award":["2023S018087"],"award-info":[{"award-number":["2023S018087"]}],"id":[{"id":"10.13039\/501100012245","id-type":"DOI","asserted-by":"crossref"}]},{"name":"Maoming Science and Technology Program","award":["MMSZX2025005"],"award-info":[{"award-number":["MMSZX2025005"]}]},{"name":"College Students\u2019 Innovation and Entrepreneurship Training Program of Guangdong Province","award":["25B115"],"award-info":[{"award-number":["25B115"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Biology"],"abstract":"<jats:p>PBU (N-phenyl-N\u2032-thiazolylurea) promotes callus induction and adventitious bud differentiation in eucalyptus, but the miRNA-mediated regulatory mechanisms underlying these effects remain unclear. In this study, calli of Eucalyptus urophylla \u00d7 E. grandis clone DH32-29 with distinct phenotypes were cultured at four PBU concentrations (0, 0.1, 1 and 5 mg L\u22121) and analyzed by small-RNA sequencing, targeted metabolomics, and qRT-PCR validation. A total of 114 common differentially expressed miRNAs were identified, targeting 610 mRNAs. Functional enrichment analysis revealed that these targets were predominantly associated with lignin metabolism, phenylpropanoid metabolism, biotin metabolism, tryptophan metabolism, protein processing in the endoplasmic reticulum, and galactose metabolism. Metabolomic profiling detected 3029 metabolites, with differential metabolites enriched in the ABC transporter pathway, galloyl sugar biosynthesis, and cofactor biosynthesis. Key miRNA families, including miR164, miR165\/166, and miR396, exhibited PBU concentration-dependent expression patterns and were predicted, based on in silico target prediction and qRT-PCR co-expression, to be potentially associated with target genes involved in lignin biosynthesis, ROS-related metabolism, and cytokinin homeostasis; these regulatory relationships remain to be experimentally validated. Among the tested concentrations, 1 mg L\u22121 PBU was the dosage associated with the strongest reprogramming of secondary metabolism and with metabolic signatures suggestive of better preserved redox homeostasis; future work will build on this reference dataset with quantitative regeneration phenotyping and direct redox measurements to confirm this candidate optimum. These findings provide new insights into PBU-mediated in vitro regeneration in eucalyptus and offer a molecular basis for optimizing regeneration systems in E. urophylla \u00d7 E. grandis. These findings provide new insights into the miRNA-metabolite regulatory network underlying phenylurea-mediated callus differentiation in woody plants.<\/jats:p>","DOI":"10.3390\/biology15151315","type":"journal-article","created":{"date-parts":[[2026,8,6]],"date-time":"2026-08-06T08:08:02Z","timestamp":1786003682000},"page":"1315","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["PBU Concentration-Dependent Regulation of Callus Differentiation in Eucalyptus urophylla \u00d7 E. grandis: Integrated miRNA and Metabolomic Insights"],"prefix":"10.3390","volume":"15","author":[{"given":"Chaohong","family":"Wang","sequence":"first","affiliation":[{"name":"College of Biology and Food Engineering, Guangdong University of Petrochemical Technology, Maoming 525000, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Taoming","family":"Yang","sequence":"additional","affiliation":[{"name":"College of Biology and Food Engineering, Guangdong University of Petrochemical Technology, Maoming 525000, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8114-5540","authenticated-orcid":false,"given":"Lejun","family":"Ouyang","sequence":"additional","affiliation":[{"name":"College of Biology and Food Engineering, Guangdong University of Petrochemical Technology, Maoming 525000, China"},{"name":"Guangdong Provincial Engineering and Technology Research Center for Gene Resource Exploration and Precision Editing of Lingnan Characteristic Plants, Maoming 525000, China"},{"name":"Guangdong Provincial Key Laboratory of Green and Intelligent Agricultural Production, Maoming 525000, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jiapeng","family":"Zeng","sequence":"additional","affiliation":[{"name":"College of Biology and Food Engineering, Guangdong University of Petrochemical Technology, Maoming 525000, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kang","family":"Xun","sequence":"additional","affiliation":[{"name":"College of Biology and Food Engineering, Guangdong University of Petrochemical Technology, Maoming 525000, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Limei","family":"Li","sequence":"additional","affiliation":[{"name":"College of Biology and Food Engineering, Guangdong University of Petrochemical Technology, Maoming 525000, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Bingwei","family":"Jiang","sequence":"additional","affiliation":[{"name":"Maoming Agricultural Science and Technology Promotion Center, Maoming 525000, China"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,8,6]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"50","DOI":"10.1111\/j.1467-7652.2010.00530.x","article-title":"Two EguCBF1 genes overexpressed in Eucalyptus display a different impact on stress tolerance and plant development","volume":"9","author":"Navarro","year":"2011","journal-title":"Plant Biotechnol. J."},{"key":"ref_2","first-page":"38","article-title":"Analysis and prospect of the driving forces for the rapid development of Eucalyptus plantations in China","volume":"37","author":"Liu","year":"2020","journal-title":"Eucalypt Sci. Technol."},{"key":"ref_3","doi-asserted-by":"crossref","unstructured":"Shen, C., Li, L.M., Ouyang, L.J., Wang, J., Xu, C., Chen, H., Jiang, B., Wu, W., Zeng, J., and Zeng, B. (2023). High-quality genome of E. urophylla \u00d7 E. grandis and comparative genomics provide insights on evolution and diversification of Eucalyptus. BMC Genom., 24.","DOI":"10.1186\/s12864-023-09318-0"},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"1229","DOI":"10.1126\/science.227.4691.1229","article-title":"A simple and general method for transferring genes into plants","volume":"227","author":"Horsch","year":"1985","journal-title":"Science"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"969","DOI":"10.1111\/j.1365-313X.2006.02836.x","article-title":"Early infection of scutellum tissue with Agrobacterium allows high-speed transformation of rice","volume":"47","author":"Toki","year":"2006","journal-title":"Plant J."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"21","DOI":"10.1080\/07352689.2012.696461","article-title":"Chrysanthemum biotechnology: Quo vadis?","volume":"32","author":"Silva","year":"2013","journal-title":"Crit. Rev. Plant Sci."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"81","DOI":"10.1007\/BF00269239","article-title":"Leaf disc transformation of cultivated tomato (L. esculentum) using Agrobacterium tumefaciens","volume":"5","author":"McCormick","year":"1986","journal-title":"Plant Cell Rep."},{"key":"ref_8","first-page":"14","article-title":"Research progress in Eucalyptus tissue culture","volume":"13","author":"Xie","year":"2000","journal-title":"World For. Res."},{"key":"ref_9","first-page":"24","article-title":"Callus induction and construction of rapid propagation system for regenerated plants of Eucalyptus 12ABL","volume":"26","author":"Tan","year":"2005","journal-title":"Chin. J. Trop. Crops"},{"key":"ref_10","first-page":"14","article-title":"Establishment of high-efficiency tissue culture regeneration system for Eucalyptus \u2018Guangzhou No. 1\u2019","volume":"40","author":"Ouyang","year":"2012","journal-title":"J. Northeast For. Univ."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"dev200879","DOI":"10.1242\/dev.200879","article-title":"Developmental trajectory of pluripotent stem cell establishment in Arabidopsis callus guided by a quiescent center-related gene network","volume":"150","author":"Zhai","year":"2023","journal-title":"Development"},{"key":"ref_12","first-page":"602","article-title":"Effects of exogenous GA3 application on xylem development in Eucalyptus grandis","volume":"16","author":"Guo","year":"2018","journal-title":"Mol. Plant Breed."},{"key":"ref_13","doi-asserted-by":"crossref","unstructured":"Chen, Y., Yu, H., Wang, Y., Li, F., Xing, Y., and Ge, X. (2022). Uniconazole augments abscisic acid in promoting somatic embryogenesis in cotton (Gossypium hirsutum L.). Front. Plant Sci., 13.","DOI":"10.3389\/fpls.2022.865778"},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"847","DOI":"10.1007\/s00442-008-1042-x","article-title":"Methyl jasmonate does not induce changes in Eucalyptus grandis leaves that alter the effect of constitutive defences on larvae of a specialist herbivore","volume":"156","author":"Henery","year":"2008","journal-title":"Oecologia"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"8984","DOI":"10.5897\/AJB11.636","article-title":"Thidiazuron: A multi-dimensional plant growth regulator","volume":"10","author":"Guo","year":"2011","journal-title":"Afr. J. Biotechnol."},{"key":"ref_16","first-page":"840","article-title":"Differential expression of miRNA396 and CKX genes in callus of Eucalyptus urophylla \u00d7 E. grandis induced by different concentrations of the novel Mitogen PBU","volume":"42","author":"Liu","year":"2022","journal-title":"Bull. Bot. Res."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"19","DOI":"10.1146\/annurev.arplant.57.032905.105218","article-title":"MicroRNAs and their regulatory roles in plants","volume":"57","author":"Bartel","year":"2006","journal-title":"Annu. Rev. Plant Biol."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"349","DOI":"10.1105\/tpc.114.135186","article-title":"An intrinsic microRNA timer regulates progressive decline in shoot regenerative capacity in plants","volume":"27","author":"Zhang","year":"2015","journal-title":"Plant Cell"},{"key":"ref_19","first-page":"214","article-title":"Genome-wide identification and analysis of TCP gene family in Populus yunnanensis","volume":"40","author":"Chen","year":"2024","journal-title":"Biotechnol. Bull."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"257","DOI":"10.1038\/nature01958","article-title":"Control of leaf morphogenesis by microRNAs","volume":"425","author":"Palatnik","year":"2003","journal-title":"Nature"},{"key":"ref_21","first-page":"4","article-title":"MicroRNA319-mediated gene regulatory network impacts leaf development and morphogenesis in poplar","volume":"1","author":"Cheng","year":"2021","journal-title":"For. Res."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"2362","DOI":"10.1111\/jipb.13759","article-title":"Coordination of miR319-TaPCF8 with TaSPL14 orchestrates auxin signaling and biosynthesis to regulate plant height in common wheat","volume":"66","author":"Han","year":"2024","journal-title":"J. Integr. Plant Biol."},{"key":"ref_23","unstructured":"Cao, D.Y. (2018). Functional Study of miR396 in Tomato Fruit Growth and Development. [Master\u2019s Thesis, China Agricultural University]."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"227","DOI":"10.5483\/BMBRep.2019.52.4.051","article-title":"Biological roles and an evolutionary sketch of the GRF-GIF transcriptional complex in plants","volume":"52","author":"Jeong","year":"2019","journal-title":"BMB Rep."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"1274","DOI":"10.1038\/s41587-020-0703-0","article-title":"A GRF\u2013GIF chimeric protein improves the regeneration efficiency of transgenic plants","volume":"38","author":"Debernardi","year":"2020","journal-title":"Nat. Biotechnol."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"1655","DOI":"10.1016\/j.cj.2024.08.003","article-title":"Gene editing and overexpression of soybean miR396a reveals its role in salinity tolerance and development","volume":"12","author":"Chen","year":"2024","journal-title":"Crop J."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"2079","DOI":"10.1111\/pbi.14326","article-title":"miR396b\/GRF6 module contributes to salt tolerance in rice","volume":"22","author":"Yuan","year":"2024","journal-title":"Plant Biotechnol. J."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"1789","DOI":"10.1093\/pcp\/pcx117","article-title":"ARGONAUTE10 inhibits in vitro shoot regeneration via repression of miR165\/166 in Arabidopsis thaliana","volume":"58","author":"Xue","year":"2017","journal-title":"Plant Cell Physiol."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"uhac192","DOI":"10.1093\/hr\/uhac192","article-title":"miR164g-MsNAC022 acts as a novel module mediating drought response by transcriptional regulation of reactive oxygen species scavenging systems in apple","volume":"9","author":"Peng","year":"2022","journal-title":"Hortic. Res."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"D155","DOI":"10.1093\/nar\/gky1141","article-title":"miRBase: From microRNA sequences to function","volume":"47","author":"Kozomara","year":"2019","journal-title":"Nucleic Acids Res."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"2521","DOI":"10.1093\/bioinformatics\/bty972","article-title":"miRDeep-P2: Accurate and fast analysis of the microRNA transcriptome in plants","volume":"35","author":"Kuang","year":"2019","journal-title":"Bioinformatics"},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"2101","DOI":"10.1093\/pcp\/pcs152","article-title":"Reference genes for high-throughput quantitative reverse transcription-PCR analysis of gene expression in organs and tissues of Eucalyptus grown in various environmental conditions","volume":"53","author":"Soler","year":"2012","journal-title":"Plant Cell Physiol."},{"key":"ref_33","doi-asserted-by":"crossref","unstructured":"Su, M., Wu, X., Wang, Z., Li, L., and Ouyang, L. (2025). Construction of an editing system for forest tree genomes based on an efficient visual screening marker in Eucalyptus urophylla \u00d7 Eucalyptus grandis. Horticulturae, 11.","DOI":"10.3390\/horticulturae11040406"},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"137","DOI":"10.1146\/annurev-arplant-050312-120043","article-title":"Classification and comparison of small RNAs from plants","volume":"64","author":"Axtell","year":"2013","journal-title":"Annu. Rev. Plant Biol."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"522","DOI":"10.1038\/nrg1379","article-title":"MicroRNAs: Small RNAs with a big role in gene regulation","volume":"5","author":"He","year":"2004","journal-title":"Nat. Rev. Genet."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"70","DOI":"10.4161\/psb.5.1.10233","article-title":"Auxin signal transduction in Arabidopsis vein formation","volume":"5","author":"Donner","year":"2010","journal-title":"Plant Signal. Behav."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"1006","DOI":"10.1093\/mp\/ssu051","article-title":"Negative feedback regulation of auxin signaling by ATHB8\/ACL5-BUD2 transcription module","volume":"7","author":"Baima","year":"2014","journal-title":"Mol. Plant"},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"482","DOI":"10.1105\/tpc.111.090894","article-title":"JUNGBRUNNEN1, a reactive oxygen species-responsive NAC transcription factor, regulates longevity in Arabidopsis","volume":"24","author":"Wu","year":"2012","journal-title":"Plant Cell"},{"key":"ref_39","first-page":"2884","article-title":"miR396\u2013MsGRFs regulate the regeneration of alfalfa callus","volume":"30","author":"Chen","year":"2022","journal-title":"Acta Agrestia Sin."},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"328","DOI":"10.1006\/bbrc.1999.0199","article-title":"Isolation of a gene encoding a glycosylated cytokinin oxidase from maize","volume":"255","author":"Morris","year":"1999","journal-title":"Biochem. Biophys. Res. Commun."},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"1112","DOI":"10.1016\/j.cj.2024.07.006","article-title":"The DUF579 proteins GhIRX15s regulate cotton fiber development by interacting with proteins involved in xylan synthesis","volume":"12","author":"Li","year":"2024","journal-title":"Crop J."},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"6869","DOI":"10.1073\/pnas.93.14.6869","article-title":"Ferulate-5-hydroxylase from Arabidopsis thaliana defines a new family of cytochrome P450-dependent monooxygenases","volume":"93","author":"Meyer","year":"1996","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"110390","DOI":"10.1016\/j.plantsci.2019.110390","article-title":"MicroRNA397b negatively regulates resistance of Malus hupehensis to Botryosphaeria dothidea by modulating MhLAC7 involved in lignin biosynthesis","volume":"292","author":"Yu","year":"2020","journal-title":"Plant Sci."},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"e2415083","DOI":"10.1002\/advs.202415083","article-title":"Cell-type specific miRNA regulatory network responses to ABA stress revealed by time series transcriptional atlases in Arabidopsis","volume":"12","author":"Gao","year":"2025","journal-title":"Adv. Sci."},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"916","DOI":"10.1093\/plphys\/kiae422","article-title":"A microRNA396b-growth regulating factor module controls castor seed size by mediating auxin synthesis","volume":"196","author":"Wang","year":"2024","journal-title":"Plant Physiol."}],"container-title":["Biology"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2079-7737\/15\/15\/1315\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,8,8]],"date-time":"2026-08-08T04:14:15Z","timestamp":1786162455000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2079-7737\/15\/15\/1315"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,8,6]]},"references-count":45,"journal-issue":{"issue":"15","published-online":{"date-parts":[[2026,8]]}},"alternative-id":["biology15151315"],"URL":"https:\/\/doi.org\/10.3390\/biology15151315","relation":{},"ISSN":["2079-7737"],"issn-type":[{"value":"2079-7737","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,8,6]]}}}