Stress memory triggered by combinatorial thermos-drought priming enhances cross-tolerance of Pinus massoniana seedlings: physio-biochemical evidence from multi-stress conditioning

Stress memory triggered by combinatorial thermos-drought priming enhances cross-tolerance of Pinus massoniana seedlings: physio-biochemical evidence from multi-stress conditioning | 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 Article Stress memory triggered by combinatorial thermos-drought priming enhances cross-tolerance of Pinus massoniana seedlings: physio-biochemical evidence from multi-stress conditioning Xiaoyu Luo, Fuhua Fan This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7306710/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 The intensification of climate change has rendered high-temperature and drought stress critical constraints on forest ecosystems. Pinus massoniana Lamb., an ecologically and economically significant conifer species renowned for its abiotic stress resilience, presents an ideal model for investigating stress memory formation in woody perennials. This study establishes a novel priming protocol involving cyclical stress exposure to systematically examine physiological memory formation and subsequent stress tolerance enhancement. Three-month-old seedlings underwent three distinct priming regiments: thermos priming (35/30℃, 14/10 photoperiod), drought priming (~ 25% field capacity), and combinatorial stress (thermos-drought), followed by a 15-day recovery phase before imposing identical combinatorial stress. Comprehensive analyses of growth dynamics, oxidative stress biomarkers, antioxidant systems, and osmoregulatory compounds were conducted through a multi-methodological approach encompassing morphometric measurements, spectrophotometric assays, and enzymatic activity quantification. Principal component analysis and membership function analysis were employed to evaluate the impact of different adversity training treatments on the tolerance of seedlings to combined stress, thereby clarifying the actual effectiveness of adversity training in enhancing the stress resistance. Results indicated that, compared with non-primed controls, those subjected to adversity priming exhibited a reduced inhibitory effect on stem elongation and root collar diameter growth under subsequent stress, along with decrease in lipid peroxidation (malondialdehyde content), reduced relative cell conductance, and enhanced antioxidant capacity. Notably, combinational priming induced synergistic osmoregulatory adaptation, elevating proline accumulation while reducing soluble sugar consumption compared to single-stress priming, suggested the most significant improvements in stress tolerance. The comprehensive evaluation index revealed a stress resilience hierarchy: combinational priming > thermos priming > drought priming > non-primed controls. This study provides the first empirical evidence of stress memory formation in P. massoniana through cross-tolerance mechanisms, demonstrating that cyclical sublethal stress exposure enhances subsequent stress tolerance. The superior efficacy of combinational priming highlights the necessity of multi-stress experimental designs to accurately simulate natural stress regimes. These findings advance our understanding of ecological memory in perennial woody plants and establish practical protocols for developing climate-resilient forestry through targeted stress hardening. Biological sciences/Physiology Biological sciences/Plant sciences Pinus massoniana High temperature Drought Combined stress Stress memory Full Text Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted 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-7306710","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":509132322,"identity":"8a362e13-024f-4619-a79f-ebb55236eea3","order_by":0,"name":"Xiaoyu Luo","email":"","orcid":"","institution":"Guizhou University","correspondingAuthor":false,"prefix":"","firstName":"Xiaoyu","middleName":"","lastName":"Luo","suffix":""},{"id":509132323,"identity":"5caaaa4c-8f84-4b6e-9f66-c6c51a3ed614","order_by":1,"name":"Fuhua Fan","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAxElEQVRIiWNgGAWjYBAC9hmMDxgYDGwgPB5itPDcYDZgYChII1nLh8OkaJFuZvxcYHDebv6MBMYHb9sY5M0JapE5zCw9w+B2cuOMBGbDuW0MhjsbCGixl8g/IM0D1MIskcAmzdvGkGBwgJAtEsnMv3kMziWzSSSw/yZWCxvQlgN2PEBbmInWYs1jkJwgwfOwWXLOOQnDDcQ47DbPHzt7+fbkgx/elNnIE7QFBhIbGBgbgLQEkeqBwJ54paNgFIyCUTDiAAAhhjZzynxSaQAAAABJRU5ErkJggg==","orcid":"","institution":"Guizhou University","correspondingAuthor":true,"prefix":"","firstName":"Fuhua","middleName":"","lastName":"Fan","suffix":""}],"badges":[],"createdAt":"2025-08-06 07:08:33","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7306710/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7306710/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":93368956,"identity":"cb0ff93d-6769-41b2-be6c-412813b71223","added_by":"auto","created_at":"2025-10-13 06:02:28","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":795868,"visible":true,"origin":"","legend":"","description":"","filename":"Manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7306710/v1_covered_0988d463-db04-4571-8349-65d8fce769a7.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Stress memory triggered by combinatorial thermos-drought priming enhances cross-tolerance of Pinus massoniana seedlings: physio-biochemical evidence from multi-stress conditioning","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":"Pinus massoniana, High temperature, Drought, Combined stress, Stress memory","lastPublishedDoi":"10.21203/rs.3.rs-7306710/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7306710/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe intensification of climate change has rendered high-temperature and drought stress critical constraints on forest ecosystems. \u003cem\u003ePinus massoniana\u003c/em\u003e Lamb., an ecologically and economically significant conifer species renowned for its abiotic stress resilience, presents an ideal model for investigating stress memory formation in woody perennials. This study establishes a novel priming protocol involving cyclical stress exposure to systematically examine physiological memory formation and subsequent stress tolerance enhancement. Three-month-old seedlings underwent three distinct priming regiments: thermos priming (35/30℃, 14/10 photoperiod), drought priming (~\u0026thinsp;25% field capacity), and combinatorial stress (thermos-drought), followed by a 15-day recovery phase before imposing identical combinatorial stress. Comprehensive analyses of growth dynamics, oxidative stress biomarkers, antioxidant systems, and osmoregulatory compounds were conducted through a multi-methodological approach encompassing morphometric measurements, spectrophotometric assays, and enzymatic activity quantification. Principal component analysis and membership function analysis were employed to evaluate the impact of different adversity training treatments on the tolerance of seedlings to combined stress, thereby clarifying the actual effectiveness of adversity training in enhancing the stress resistance. Results indicated that, compared with non-primed controls, those subjected to adversity priming exhibited a reduced inhibitory effect on stem elongation and root collar diameter growth under subsequent stress, along with decrease in lipid peroxidation (malondialdehyde content), reduced relative cell conductance, and enhanced antioxidant capacity. Notably, combinational priming induced synergistic osmoregulatory adaptation, elevating proline accumulation while reducing soluble sugar consumption compared to single-stress priming, suggested the most significant improvements in stress tolerance. The comprehensive evaluation index revealed a stress resilience hierarchy: combinational priming\u0026thinsp;\u0026gt;\u0026thinsp;thermos priming\u0026thinsp;\u0026gt;\u0026thinsp;drought priming\u0026thinsp;\u0026gt;\u0026thinsp;non-primed controls. This study provides the first empirical evidence of stress memory formation in \u003cem\u003eP. massoniana\u003c/em\u003e through cross-tolerance mechanisms, demonstrating that cyclical sublethal stress exposure enhances subsequent stress tolerance. The superior efficacy of combinational priming highlights the necessity of multi-stress experimental designs to accurately simulate natural stress regimes. These findings advance our understanding of ecological memory in perennial woody plants and establish practical protocols for developing climate-resilient forestry through targeted stress hardening.\u003c/p\u003e","manuscriptTitle":"Stress memory triggered by combinatorial thermos-drought priming enhances cross-tolerance of Pinus massoniana seedlings: physio-biochemical evidence from multi-stress conditioning","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-03 08:13:09","doi":"10.21203/rs.3.rs-7306710/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":"2858b32a-f94e-47d2-9191-b3a9ab5e5c8e","owner":[],"postedDate":"September 3rd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":54073107,"name":"Biological sciences/Physiology"},{"id":54073108,"name":"Biological sciences/Plant sciences"}],"tags":[],"updatedAt":"2025-10-13T05:54:20+00:00","versionOfRecord":[],"versionCreatedAt":"2025-09-03 08:13:09","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7306710","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7306710","identity":"rs-7306710","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}