A multi-layered systems biology framework reveals dual-phased regulators and hormonal crosstalk underlying soybean cold tolerance

A multi-layered systems biology framework reveals dual-phased regulators and hormonal crosstalk underlying soybean cold tolerance | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article A multi-layered systems biology framework reveals dual-phased regulators and hormonal crosstalk underlying soybean cold tolerance Hao-Yu Liu, Pei-Hsiu Kao, Supaporn Baiya, Chung-Feng Kao This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6971483/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 05 Nov, 2025 Read the published version in Plant Cell Reports → Version 1 posted 5 You are reading this latest preprint version Abstract Cold stress poses a significant threat to soybean ( Glycine max (L.) Merr) productivity, particularly during early developmental stages. Traditional approaches for identifying cold-responsive genes have been limited by gene size bias, pathway redundancy, and lack of integrative validation. To address these challenges, we developed a multi-layered systems biology framework, termed SNFE (systems and network-based feature engineering), designed to uncover key cold-tolerant genes (CTgenes) by leveraging both omics and non-omics data in a network-informed context. The SNFE framework integrates five analytical layers: functional pathway enrichment, pathway crosstalk, co-functional network construction, network topology analysis, and experimental validation. From an initial pool of cold-responsive genes, SNFE identified 10 key CTgenes that demonstrated high connectivity, regulatory importance, and consistent differential expression in short- and mid-term cold conditions. These genes were validated via independent transcriptomic datasets, Quantitative real-time PCR analysis, and hormone profiling. Notably, SNFE revealed novel regulatory mechanisms, including dual-timed transcription factors, ABA–JA hormone synergy in membrane stabilization, and convergence of abiotic and biotic stress signaling. A Sankey diagram and volcano plot further confirmed that most CTgenes reside at key regulatory nodes, linking upstream functions to downstream cold-tolerance pathways. SNFE is a reliable, efficient, and interpretable tool that not only improves prediction accuracy but also enables the discovery of novel biological insights. Its scalability and analytical depth make it a powerful platform for dissecting complex stress responses in crops. This framework provides a strategic foundation for molecular breeding programs aiming to enhance climate resilience in soybean and other crops. soybean (Glycine max) multi-layered systems biology systems and network-based feature engineering (SNFE) key CTgenes discovery gene-level statistic hormonal crosstalk dual-phased regulators Full Text Supplementary Files FigureS1.pdf TableS1.docx TableS2Pathwayenrichmentanalysis.xlsx TableS3Networktopologyanalysis.xlsx TableS4CTgenescandidatesKaoetal.2022.xlsx TableS5Validationsamplesoybeanleafcoldtrtraw.xlsx TableS6SequenceoftheprimersqRTPCRofkeyCTgenes.xlsx Cite Share Download PDF Status: Published Journal Publication published 05 Nov, 2025 Read the published version in Plant Cell Reports → Version 1 posted Editorial decision: Minor revisions 23 Sep, 2025 Reviewers agreed at journal 31 Jul, 2025 Reviewers invited by journal 21 Jul, 2025 Editor assigned by journal 21 Jul, 2025 First submitted to journal 18 Jul, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6971483","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":488831330,"identity":"a216f23d-0882-4eff-9435-ace5f3e2f40e","order_by":0,"name":"Hao-Yu Liu","email":"","orcid":"","institution":"National Chung Hsing University","correspondingAuthor":false,"prefix":"","firstName":"Hao-Yu","middleName":"","lastName":"Liu","suffix":""},{"id":488831331,"identity":"493d9da0-bbb3-41c7-bf1c-fe842857eabf","order_by":1,"name":"Pei-Hsiu Kao","email":"","orcid":"","institution":"The University of 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