RcDHD/SDH gene family differentially drive gallic and ellagic acid biosynthesis in Rubus chingii fruit: insights from transient overexpression and metabolic profiling

RcDHD/SDH gene family differentially drive gallic and ellagic acid biosynthesis in Rubus chingii fruit: insights from transient overexpression and metabolic profiling | 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 RcDHD/SDH gene family differentially drive gallic and ellagic acid biosynthesis in Rubus chingii fruit: insights from transient overexpression and metabolic profiling Xueli An, Yuezhen Li, Dongfeng Yang, Jianhong Chen, Ling Jiang, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8341597/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 3-dehydroquinic acid dehydrogenase/shikimate dehydrogenase ( DHD/SDH ) gene, which encodes a protein, acts as a dual-enzyme capable of catalyzing both shikimate and gallic acid synthesis. This dual-enzyme is playing a pivotal regulatory role in the gallic acid metabolic pathway. Objective: To investigate the functional roles of DHD/SDH in the gallic acid and ellagic acid synthesis pathways within the fruit of Chinese raspberry ( Rubus chingii Hu). Method: Using Rubus chingii Hu ( R. chingii ) as experimental material, Agrobacterium-mediated transformation was utilized to introduce over-expression vectors carrying the target gene. Specifically, Agrobacterium cultures harboring the R. chingii RcDHD/SDH target gene over-expression vectors ranging from pCAMBIA1300-RcDHD/SDH1 to RcDHD/SDH4-GFP were injected into raspberry fruit tissue for transient expression. To evaluate the impact, fluorescent quantitative PCR was employed to assess DHD/SDH gene expression levels, while HPLC was used to determine gallic acid and ellagic acid content, providing preliminary insights into the biological roles of the RcDHD/SDH gene family. Results: Subcellular localization experiments revealed that these gene products are localized to the nucleus. This gene cluster is pivotal in controlling the synthesis of phenolic acid in R. chingii through temporal expression patterns, with its activity aligning closely with the level of ellagic acid and gallic acid. In the case of thornless cultivars, RcDHD/SDH1 - RcDHD/SDH2 take center stage, enabling an early rapid accumulation, whereas thorny cultivars activate RcDHD/SDH3 - RcDHD/SDH4 to implement a sustained synthesis approach during late-stage, highlighting gene functional differentiation in secondary metabolic strategies between cultivars. Conclusion: RcDHD/SDH stimulates biosynthesis and accumulation of gallic and ellagic acid in R. chingii , providing a theoretical basis for molecular breeding to enhance fruit ellagic acid content. Biological sciences/Biochemistry Biological sciences/Biological techniques Biological sciences/Biotechnology Biological sciences/Molecular biology Biological sciences/Plant sciences Rubus chingii Hu gallic acid ellagic acid RcDHD/SDH gene family transient expression 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-8341597","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":564822736,"identity":"ff1cecb3-da1d-4e82-b7ec-8bcd67c0698e","order_by":0,"name":"Xueli An","email":"","orcid":"","institution":"Zhejiang Sci-Tech University","correspondingAuthor":false,"prefix":"","firstName":"Xueli","middleName":"","lastName":"An","suffix":""},{"id":564822739,"identity":"4298cca2-6994-4b99-8065-0a4acc54de17","order_by":1,"name":"Yuezhen Li","email":"","orcid":"","institution":"Zhejiang Sci-Tech 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Rubus chingii fruit: insights from transient overexpression and metabolic profiling","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":"Rubus chingii Hu, gallic acid, ellagic acid, RcDHD/SDH gene family, transient expression","lastPublishedDoi":"10.21203/rs.3.rs-8341597/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8341597/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe 3-dehydroquinic acid dehydrogenase/shikimate dehydrogenase (\u003cem\u003eDHD/SDH\u003c/em\u003e) gene, which encodes a protein, acts as a dual-enzyme capable of catalyzing both shikimate and gallic acid synthesis. This dual-enzyme is playing a pivotal regulatory role in the gallic acid metabolic pathway.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eObjective: \u003c/strong\u003eTo investigate the functional roles of \u003cem\u003eDHD/SDH\u003c/em\u003e in the gallic acid and ellagic acid synthesis pathways within the fruit of Chinese raspberry (\u003cem\u003eRubus chingii\u003c/em\u003e Hu).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethod: \u003c/strong\u003eUsing \u003cem\u003eRubus chingii\u003c/em\u003e Hu (\u003cem\u003eR. chingii\u003c/em\u003e) as experimental material, Agrobacterium-mediated transformation was utilized to introduce over-expression vectors carrying the target gene. Specifically, Agrobacterium cultures harboring the \u003cem\u003eR. chingii\u003c/em\u003e \u003cem\u003eRcDHD/SDH\u003c/em\u003etarget gene over-expression vectors ranging from pCAMBIA1300-RcDHD/SDH1 to RcDHD/SDH4-GFP were injected into raspberry fruit tissue for transient expression. To evaluate the impact, fluorescent quantitative PCR was employed to assess \u003cem\u003eDHD/SDH\u003c/em\u003e gene expression levels, while HPLC was used to determine gallic acid and ellagic acid content, providing preliminary insights into the biological roles of the \u003cem\u003eRcDHD/SDH \u003c/em\u003egene family.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults: \u003c/strong\u003eSubcellular localization experiments revealed that these gene products are localized to the nucleus. This\u003cem\u003e \u003c/em\u003egene cluster is pivotal in controlling the synthesis of phenolic acid in \u003cem\u003eR. chingii\u003c/em\u003e through temporal expression patterns, with its activity aligning closely with the level of ellagic acid and gallic acid. In the case of thornless cultivars, \u003cem\u003eRcDHD/SDH1\u003c/em\u003e-\u003cem\u003eRcDHD/SDH2\u003c/em\u003e take center stage, enabling an early rapid accumulation, whereas thorny cultivars activate\u003cem\u003eRcDHD/SDH3\u003c/em\u003e-\u003cem\u003eRcDHD/SDH4\u003c/em\u003e to implement a sustained synthesis approach during late-stage, highlighting gene functional differentiation in secondary metabolic strategies between cultivars.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion:\u003c/strong\u003e \u003cem\u003eRcDHD/SDH\u003c/em\u003estimulates biosynthesis and accumulation of gallic and ellagic acid in \u003cem\u003eR. chingii\u003c/em\u003e, providing a theoretical basis for molecular breeding to enhance fruit ellagic acid content.\u003c/p\u003e","manuscriptTitle":"RcDHD/SDH gene family differentially drive gallic and ellagic acid biosynthesis in Rubus chingii fruit: insights from transient overexpression and metabolic profiling","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-24 12:17:21","doi":"10.21203/rs.3.rs-8341597/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":"1b7f918d-6783-4781-a54d-c5ad5c771d83","owner":[],"postedDate":"December 24th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":60138507,"name":"Biological sciences/Biochemistry"},{"id":60138508,"name":"Biological sciences/Biological techniques"},{"id":60138509,"name":"Biological sciences/Biotechnology"},{"id":60138510,"name":"Biological sciences/Molecular biology"},{"id":60138511,"name":"Biological sciences/Plant sciences"}],"tags":[],"updatedAt":"2026-01-08T05:39:50+00:00","versionOfRecord":[],"versionCreatedAt":"2025-12-24 12:17:21","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8341597","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8341597","identity":"rs-8341597","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}