The alfalfa AP2/ERF transcription factor MsCBF4 enhances cold tolerance in Arabidopsis thaliana

The alfalfa AP2/ERF transcription factor MsCBF4 enhances cold tolerance in Arabidopsis thaliana | 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 The alfalfa AP2/ERF transcription factor MsCBF4 enhances cold tolerance in Arabidopsis thaliana Naipeng Ren, Hui Zang, Yifu Xuan, Jielin Liu, Xiangping Liu, Guoliang Li This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7734621/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 24 Nov, 2025 Read the published version in BMC Plant Biology → Version 1 posted 19 You are reading this latest preprint version Abstract Background Alfalfa ( Medicago sativa L.) is globally important forage crop. The survival and consequently the productivity of this leguminous crop is determined by the capacity of the plant to tolerate cold stress, particularly at higher latitudes. However, the molecular mechanisms of how alfalfa defends against cold stress remain unclear. Results In this study, we cloned the candidate gene MsCBF4 from the cold-tolerant alfalfa genotype “Dongnong NO.1”. This gene belongs to the APETALA2/Ethylene response factor (AP2/ERF) family of transcription factors (TFs), which serve as transcriptional activators with a transcription activation domain located at the C-terminal. The protein encoded by MsCBF4 is localized in the cell nucleus. MsCBF4 is induced by cold and expressed in roots, stems, leaves, flowers, and pods, with high expression levels in root and leaf tissues. Arabidopsis thaliana lines that overexpress MsCBF4 demonstrate enhanced cold tolerance during both the seedling and mature stages, and the expression of the gene is thought to improve the physiological scavenging of reactive oxygen species (ROSs), inhibiting cell membrane damage. Furthermore, under freezing stress, the expression levels of key genes involved in abiotic stress resistance are upregulated in transgenic Arabidopsis thaliana . Herein, we describe MsCBF4 is positively regulated by MsERF6 . Conclusion These findings suggest that MsCBF4 can enhance cold tolerance in Arabidopsis thaliana by alleviating oxidative stress, protecting membrane integrity, and upregulating stress-responsive genes. Alfalfa MsCBF4 Cold stress Antioxidant system Arabidopsis thaliana Full Text Additional Declarations No competing interests reported. Supplementary Files Supplementarymaterial.zip Cite Share Download PDF Status: Published Journal Publication published 24 Nov, 2025 Read the published version in BMC Plant Biology → Version 1 posted Editorial decision: Revision requested 27 Oct, 2025 Reviews received at journal 27 Oct, 2025 Reviews received at journal 22 Oct, 2025 Reviewers agreed at journal 21 Oct, 2025 Reviews received at journal 20 Oct, 2025 Reviewers agreed at journal 19 Oct, 2025 Reviewers agreed at journal 18 Oct, 2025 Reviewers agreed at journal 16 Oct, 2025 Reviewers agreed at journal 16 Oct, 2025 Reviewers agreed at journal 16 Oct, 2025 Reviewers agreed at journal 15 Oct, 2025 Reviewers agreed at journal 15 Oct, 2025 Reviewers agreed at journal 15 Oct, 2025 Reviewers agreed at journal 15 Oct, 2025 Reviewers invited by journal 15 Oct, 2025 Editor assigned by journal 10 Oct, 2025 Editor invited by journal 09 Oct, 2025 Submission checks completed at journal 07 Oct, 2025 First submitted to journal 07 Oct, 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. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-7734621","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":535376165,"identity":"0a3354b2-cfb1-4b57-b2ed-b4ccfc281e62","order_by":0,"name":"Naipeng Ren","email":"","orcid":"","institution":"College of Agriculture, Heilongjiang Bayi Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Naipeng","middleName":"","lastName":"Ren","suffix":""},{"id":535376166,"identity":"7d6ed124-7352-431b-b110-1a259b3866c5","order_by":1,"name":"Hui Zang","email":"","orcid":"","institution":"College of Animal Science and 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thaliana","fulltext":[],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":false,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":true,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":true,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-plant-biology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pbio","sideBox":"Learn more about [BMC Plant Biology](http://bmcplantbiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/pbio/default.aspx","title":"BMC Plant Biology","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Alfalfa, MsCBF4, Cold stress, Antioxidant system, Arabidopsis thaliana","lastPublishedDoi":"10.21203/rs.3.rs-7734621/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7734621/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e\u003cp\u003eAlfalfa (\u003cem\u003eMedicago sativa\u003c/em\u003e L.) is globally important forage crop. The survival and consequently the productivity of this leguminous crop is determined by the capacity of the plant to tolerate cold stress, particularly at higher latitudes. However, the molecular mechanisms of how alfalfa defends against cold stress remain unclear.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003eIn this study, we cloned the candidate gene \u003cem\u003eMsCBF4\u003c/em\u003e from the cold-tolerant alfalfa genotype \u0026ldquo;Dongnong NO.1\u0026rdquo;. This gene belongs to the APETALA2/Ethylene response factor (AP2/ERF) family of transcription factors (TFs), which serve as transcriptional activators with a transcription activation domain located at the C-terminal. The protein encoded by \u003cem\u003eMsCBF4\u003c/em\u003e is localized in the cell nucleus. \u003cem\u003eMsCBF4\u003c/em\u003e is induced by cold and expressed in roots, stems, leaves, flowers, and pods, with high expression levels in root and leaf tissues. \u003cem\u003eArabidopsis thaliana\u003c/em\u003e lines that overexpress \u003cem\u003eMsCBF4\u003c/em\u003e demonstrate enhanced cold tolerance during both the seedling and mature stages, and the expression of the gene is thought to improve the physiological scavenging of reactive oxygen species (ROSs), inhibiting cell membrane damage. Furthermore, under freezing stress, the expression levels of key genes involved in abiotic stress resistance are upregulated in transgenic \u003cem\u003eArabidopsis thaliana\u003c/em\u003e. Herein, we describe \u003cem\u003eMsCBF4\u003c/em\u003e is positively regulated by \u003cem\u003eMsERF6\u003c/em\u003e.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e\u003cp\u003eThese findings suggest that \u003cem\u003eMsCBF4\u003c/em\u003e can enhance cold tolerance in \u003cem\u003eArabidopsis thaliana\u003c/em\u003e by alleviating oxidative stress, protecting membrane integrity, and upregulating stress-responsive genes.\u003c/p\u003e","manuscriptTitle":"The alfalfa AP2/ERF transcription factor MsCBF4 enhances cold tolerance in Arabidopsis thaliana","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-29 19:36:41","doi":"10.21203/rs.3.rs-7734621/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-10-27T07:51:16+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-27T07:30:59+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-23T03:43:08+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"200231169210141931012501202549287476026","date":"2025-10-21T04:51:38+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-20T07:04:53+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"260723116973163650088011540220667630478","date":"2025-10-19T05:58:23+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"74807882814334041000687038850981289843","date":"2025-10-18T07:33:07+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"163943951724030106939709225620567616321","date":"2025-10-17T00:54:36+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"253623230351491530632489895411461699532","date":"2025-10-16T13:13:23+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"289424267393350828819291122600895465017","date":"2025-10-16T07:20:17+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"286817041764664327818992643769689192592","date":"2025-10-16T01:54:51+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"154266483836164214768439941270113957399","date":"2025-10-16T01:34:12+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"130322016541606774353240591769675518050","date":"2025-10-16T01:30:19+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"213274705184195260063816358505830185684","date":"2025-10-16T01:28:42+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-10-16T01:19:27+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-10-10T17:00:19+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-10-09T15:37:19+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-10-08T02:34:33+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Plant Biology","date":"2025-10-08T02:30:32+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-plant-biology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pbio","sideBox":"Learn more about [BMC Plant Biology](http://bmcplantbiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/pbio/default.aspx","title":"BMC Plant Biology","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"b64755cd-505c-4aaa-8578-0dd49727f14c","owner":[],"postedDate":"October 29th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-12-01T16:16:23+00:00","versionOfRecord":{"articleIdentity":"rs-7734621","link":"https://doi.org/10.1186/s12870-025-07778-y","journal":{"identity":"bmc-plant-biology","isVorOnly":false,"title":"BMC Plant Biology"},"publishedOn":"2025-11-24 15:58:03","publishedOnDateReadable":"November 24th, 2025"},"versionCreatedAt":"2025-10-29 19:36:41","video":"","vorDoi":"10.1186/s12870-025-07778-y","vorDoiUrl":"https://doi.org/10.1186/s12870-025-07778-y","workflowStages":[]},"version":"v1","identity":"rs-7734621","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7734621","identity":"rs-7734621","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}