Integrated analysis of antioxidant responses, phenolic profiles and Phytohormonal changes in Thymus daenensis treated with Paclobutrazol under in vitro conditions

Integrated analysis of antioxidant responses, phenolic profiles and Phytohormonal changes in Thymus daenensis treated with Paclobutrazol under in vitro conditions | 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 Integrated analysis of antioxidant responses, phenolic profiles and Phytohormonal changes in Thymus daenensis treated with Paclobutrazol under in vitro conditions Ensiyeh Shahroudi, Fatemeh Zarinkamar This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7279797/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Paclobutrazol (PBZ), a triazole-type plant growth regulator, is widely used to modulate plant growth and stress tolerance by influencing phytohormonal signaling and oxidative metabolism. However, its effects on medicinal plants, particularly Thymus daenensis , under in vitro conditions remain largely unexplored. This study aimed to investigate the impact of different concentrations of PBZ (0, 2.5, 5, and 10 mg/L) on growth traits, antioxidant defense, phenolic metabolism, oxidative stress markers, and endogenous phytohormones in T. daenensis cultured in vitro . Results PBZ treatments induced pronounced changes in growth performance. The fresh weight of shoots progressively decreased with increasing PBZ concentrations, reaching 46% below the control at 10 mg/L, whereas root fresh weight markedly increased by 66% and 330% at 2.5 and 10 mg L⁻¹, respectively. Shoot length remained unaffected at 2.5 mg/L but declined significantly at 5 and 10 mg/L, while root length increased at 2.5 mg L⁻¹ but decreased at higher doses, with the strongest reduction at 10 mg/L. Consequently, the shoot-to-root fresh weight ratio rose at 2.5 and peaked at 5 mg/L, but declined at 10 mg/L, indicating a biomass allocation shift under elevated PBZ levels. In parallel, PBZ significantly modulated enzymatic antioxidant activities, with the greatest enhancement at 5 mg/L, and stimulated phenylalanine ammonia-lyase (PAL) and tyrosine ammonia-lyase (TAL) at moderate concentrations. Hydrogen peroxide (H₂O₂) and malondialdehyde (MDA) contents declined at 2.5 and 5 mg/L, suggesting reduced oxidative damage, while DPPH radical scavenging capacity and total flavonoid content peaked at 5 mg/L but decreased at 10 mg/L. Hormonal analysis revealed a concentration-dependent reduction in gibberellic acid (GA), indole-3-acetic acid (IAA), and brassinosteroids (BR), with the lowest values at 10 mg/L, whereas abscisic acid (ABA) progressively increased, reaching its maximum at the highest PBZ concentration. Conclusion These findings demonstrate that PBZ exerts a dose-dependent modulation of growth, antioxidant metabolism, and hormonal balance in T. daenensis under in vitro conditions. Moderate concentrations (particularly 5 mg/L) enhance antioxidant capacity, phenolic metabolism, and biomass allocation toward roots, while higher doses suppress shoot growth and accentuate hormonal shifts (reduced GA, IAA, BR; elevated ABA). Overall, PBZ shows promise as an elicitor to improve physiological resilience and potentially the medicinal quality of T. daenensis during micropropagation. Thymus daenensis paclobutrazol antioxidant defense system oxidative stress in vitro culture Full Text Additional Declarations No competing interests reported. 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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-7279797","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":502350220,"identity":"75a94e48-bca2-4c26-a9a9-17a74315d7bb","order_by":0,"name":"Ensiyeh Shahroudi","email":"","orcid":"","institution":"Tarbiat Modares University","correspondingAuthor":false,"prefix":"","firstName":"Ensiyeh","middleName":"","lastName":"Shahroudi","suffix":""},{"id":502350221,"identity":"4337712c-fb73-4e7e-a1a0-d53080d627fa","order_by":1,"name":"Fatemeh Zarinkamar","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA+0lEQVRIiWNgGAWjYJCCA0AIBMxAkg0uKEGMFrYE4rUwQLTwGCBrwQ3k288+PFxwxi6af/aZrxs+lNnl8/MfYPzwg8EiH5cWxp50g8MzbiTnzjiXu+3mjHPJljNnJDBL9jBIWDbg0MLMkMZwmOcDc27DGd5tt3nbmA0MbjAwSAP9YoDLFjb+ZyAt9bnzz/A8A2qpN7A/f4D5Nz4tPBIgW24czt1whocNqOWwgQFDAhteWyQkQLacOZ678QybGdAvxw0kbiS2WfYY4NYi35/G/JnnWHXuvDPMz258KKs24O8/fPjGj4o6nFqwAcYGBgaSNIyCUTAKRsEoQAcAz4pVaNG0s+8AAAAASUVORK5CYII=","orcid":"","institution":"Tarbiat Modares University","correspondingAuthor":true,"prefix":"","firstName":"Fatemeh","middleName":"","lastName":"Zarinkamar","suffix":""}],"badges":[],"createdAt":"2025-08-02 17:53:19","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7279797/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7279797/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":90788223,"identity":"c0f00ac6-0d00-4fcd-bc11-426a6a81c6f3","added_by":"auto","created_at":"2025-09-08 07:48:09","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":490601,"visible":true,"origin":"","legend":"","description":"","filename":"EnsiyehShahroudi.PBZ.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7279797/v1_covered_2965ee19-86f2-4e99-86a8-480c9ce9a424.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Integrated analysis of antioxidant responses, phenolic profiles and Phytohormonal changes in Thymus daenensis treated with Paclobutrazol under in vitro conditions","fulltext":[],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":false,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":true,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":true,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Thymus daenensis, paclobutrazol, antioxidant defense system, oxidative stress, in vitro culture","lastPublishedDoi":"10.21203/rs.3.rs-7279797/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7279797/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground \u003c/strong\u003ePaclobutrazol (PBZ), a triazole-type plant growth regulator, is widely used to modulate plant growth and stress tolerance by influencing phytohormonal signaling and oxidative metabolism. However, its effects on medicinal plants, particularly \u003cem\u003eThymus daenensis\u003c/em\u003e, under \u003cem\u003ein vitro\u003c/em\u003e conditions remain largely unexplored. This study aimed to investigate the impact of different concentrations of PBZ (0, 2.5, 5, and 10 mg/L) on growth traits, antioxidant defense, phenolic metabolism, oxidative stress markers, and endogenous phytohormones in \u003cem\u003eT. daenensis\u003c/em\u003e cultured \u003cem\u003ein vitro\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults \u003c/strong\u003ePBZ treatments induced pronounced changes in growth performance. The fresh weight of shoots progressively decreased with increasing PBZ concentrations, reaching 46% below the control at 10 mg/L, whereas root fresh weight markedly increased by 66% and 330% at 2.5 and 10 mg L⁻¹, respectively. Shoot length remained unaffected at 2.5 mg/L but declined significantly at 5 and 10 mg/L, while root length increased at 2.5 mg L⁻¹ but decreased at higher doses, with the strongest reduction at 10 mg/L. Consequently, the shoot-to-root fresh weight ratio rose at 2.5 and peaked at 5 mg/L, but declined at 10 mg/L, indicating a biomass allocation shift under elevated PBZ levels.\u003c/p\u003e\n\u003cp\u003eIn parallel, PBZ significantly modulated enzymatic antioxidant activities, with the greatest enhancement at 5 mg/L, and stimulated phenylalanine ammonia-lyase (PAL) and tyrosine ammonia-lyase (TAL) at moderate concentrations. Hydrogen peroxide (H₂O₂) and malondialdehyde (MDA) contents declined at 2.5 and 5 mg/L, suggesting reduced oxidative damage, while DPPH radical scavenging capacity and total flavonoid content peaked at 5 mg/L but decreased at 10 mg/L. Hormonal analysis revealed a concentration-dependent reduction in gibberellic acid (GA), indole-3-acetic acid (IAA), and brassinosteroids (BR), with the lowest values at 10 mg/L, whereas abscisic acid (ABA) progressively increased, reaching its maximum at the highest PBZ concentration.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion \u003c/strong\u003eThese findings demonstrate that PBZ exerts a dose-dependent modulation of growth, antioxidant metabolism, and hormonal balance in \u003cem\u003eT. daenensis\u003c/em\u003e under in vitro conditions. Moderate concentrations (particularly 5 mg/L) enhance antioxidant capacity, phenolic metabolism, and biomass allocation toward roots, while higher doses suppress shoot growth and accentuate hormonal shifts (reduced GA, IAA, BR; elevated ABA). Overall, PBZ shows promise as an elicitor to improve physiological resilience and potentially the medicinal quality of \u003cem\u003eT. daenensis\u003c/em\u003e during micropropagation.\u003c/p\u003e","manuscriptTitle":"Integrated analysis of antioxidant responses, phenolic profiles and Phytohormonal changes in Thymus daenensis treated with Paclobutrazol under in vitro conditions","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-08-21 12:10:14","doi":"10.21203/rs.3.rs-7279797/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"0b0a482a-05d6-4762-ba5a-a11cb94e71a9","owner":[],"postedDate":"August 21st, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-09-08T07:39:43+00:00","versionOfRecord":[],"versionCreatedAt":"2025-08-21 12:10:14","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7279797","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7279797","identity":"rs-7279797","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}