Gametophytic degradation of CENH3 - a synthetic biology approach for haploid induction in plants

Gametophytic degradation of CENH3 - a synthetic biology approach for haploid induction in plants | 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 Letter Gametophytic degradation of CENH3 - a synthetic biology approach for haploid induction in plants Andreas Houben, Saravanakumar Somasundaram, Jörg Fuchs, Maria Cuacos, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5974840/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 Double haploid technology is a critical tool for accelerating plant breeding. The manipulation of centromere-specific histone H3 (CENH3) has previously been shown to induce haploid progeny in both the model plant Arabidopsis and certain crop species. However, a universally applicable strategy for CENH3-based haploid induction across diverse crop species is still lacking. Here, we demonstrate that extreme asymmetry in centromeric CENH3 levels between parental chromosomes is a causal factor for eliminating chromosomes with ‘weaker’ centromeres during early embryogenesis. Consequently, engineered E3-ligases were utilized to selectively degrade CENH3 in the egg cell to induce wild-type paternal haploids. Also, we attempted to optimize the CENH3 degradation using E3-ligases of plant-origin. Our findings provide valuable insights to enhance the haploid induction efficiency further. The gametophytic CENH3 degradation approach leverages engineering principles and represents a potentially universal strategy for engineering CENH3-based haploid inducers in crop plants. Biological sciences/Plant sciences/Plant biotechnology/Molecular engineering in plants Biological sciences/Biological techniques/Molecular engineering/Synthetic biology Biological sciences/Plant sciences/Plant reproduction Biological sciences/Plant sciences/Plant genetics/Polyploidy in plants Figures Figure 1 Figure 2 Figure 3 Full Text Additional Declarations There is NO Competing Interest. Supplementary Files ExtendedFigures.pdf 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. 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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-5974840","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Letter","associatedPublications":[],"authors":[{"id":412580350,"identity":"188f5eb3-765a-4b08-9474-1a8da409e440","order_by":0,"name":"Andreas Houben","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABD0lEQVRIiWNgGAWjYBACAwYGNiBlw8PAAxNiB4swMDbg15IG1ZIAxMzEaTnMQLwWc/Yeswcfd5yXMec5wPzi5w+bfPlm5mNAEQbZfhxaLHvOmBvOPHObx7K3gc2yJyHNcsNhtnSgCIPxTBzWGNzIMZPmbbvNY3Ce/5sBT8JhAwNmHpAIQ+KGA3i0/G07B9TCwGb4J+G/gXwz/zegCEPifnxaGNsO8BicbWB+zJNwwIDhMA8bUARoCw6/GJw5Vm7Y25bMY9lzgI1ZJi3ZwOAwmzlQRMJ4Bi5bjjdve/Czzc7enCeB+eMbGzsD+fbmZ0ARG9l+HN5H6AVGkAQSXwKnSmQtzB8IKxsFo2AUjIKRCABUeVjG0c4iigAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0003-3419-239X","institution":"Leibniz Institute of Plant Genetics and Crop Plant Research (IPK)","correspondingAuthor":true,"prefix":"","firstName":"Andreas","middleName":"","lastName":"Houben","suffix":""},{"id":412580351,"identity":"3681f7ee-fd72-4b84-992a-17d01b1fc5b2","order_by":1,"name":"Saravanakumar Somasundaram","email":"","orcid":"","institution":"Leibniz Institute of Plant Genetics and Crop Plant Research (IPK)","correspondingAuthor":false,"prefix":"","firstName":"Saravanakumar","middleName":"","lastName":"Somasundaram","suffix":""},{"id":412580352,"identity":"940cc20c-9817-4609-8721-34e5485448bd","order_by":2,"name":"Jörg Fuchs","email":"","orcid":"https://orcid.org/0000-0003-4171-5371","institution":"IPK","correspondingAuthor":false,"prefix":"","firstName":"Jörg","middleName":"","lastName":"Fuchs","suffix":""},{"id":412580353,"identity":"a483dc64-6726-4959-ae7d-07a8399a218f","order_by":3,"name":"Maria Cuacos","email":"","orcid":"https://orcid.org/0000-0003-4910-7311","institution":"Institute of Plant Genetics and Crop Plant Research","correspondingAuthor":false,"prefix":"","firstName":"Maria","middleName":"","lastName":"Cuacos","suffix":""},{"id":412580354,"identity":"c3f79444-443c-412d-9fe7-c97ced747f4a","order_by":4,"name":"Oda Weiß","email":"","orcid":"","institution":"IPK","correspondingAuthor":false,"prefix":"","firstName":"Oda","middleName":"","lastName":"Weiß","suffix":""},{"id":412580355,"identity":"ebd0e42b-5d9f-408e-bad6-ab1fabbe059f","order_by":5,"name":"Andriy Kochevenko","email":"","orcid":"","institution":"Leibniz Institute of Plant Genetics and Crop Plant Research (IPK)","correspondingAuthor":false,"prefix":"","firstName":"Andriy","middleName":"","lastName":"Kochevenko","suffix":""},{"id":412580356,"identity":"6dc53d23-44f0-487d-b569-3c49d28bd38f","order_by":6,"name":"Jonathan Lamb","email":"","orcid":"","institution":"Division of Biological Sciences, University of Missouri, 311 Tucker Hall, Columbia, MO 65211, USA","correspondingAuthor":false,"prefix":"","firstName":"Jonathan","middleName":"","lastName":"Lamb","suffix":""}],"badges":[],"createdAt":"2025-02-06 15:25:40","currentVersionCode":1,"declarations":{"humanSubjects":false,"vertebrateSubjects":false,"conflictsOfInterestStatement":false,"humanSubjectEthicalGuidelines":false,"humanSubjectConsent":false,"humanSubjectClinicalTrial":false,"humanSubjectCaseReport":false,"vertebrateSubjectEthicalGuidelines":false},"doi":"10.21203/rs.3.rs-5974840/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5974840/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":76076472,"identity":"d50b695a-a188-4de3-9f92-a4dde44a57b1","added_by":"auto","created_at":"2025-02-12 05:40:50","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":272739,"visible":true,"origin":"","legend":"\u003cp\u003eCENH3 asymmetry and haploid induction. A) Difference between male and female gametes of [cenh3(-/-), EYFP-gCENH3(+/+)] in terms of detectable EYFP-CENH3 signals. (n) indicates the number of pollen/ovules analyzed. B) EYFP-CENH3 (green) is present in sperm nuclei but absent in the vegetative nucleus (top). Boxed region is further enlarged. Nuclei were stained with DAPI (blue). S - sperm nuclei, V - vegetative nucleus. An egg cell labeled with histone H2B-tdTomato (red) which shows no detectable EYFP-CENH3 signals (bottom). Boxed region is further enlarged. C-G) Each row represents different cross combinations for which the parents are indicated in the first two columns. Column 3: EYFP-CENH3 (green) signals of a representative apical-cell nuclei of one-cell embryo is shown. Column 4: A bar plot showing the number of EYFP-CENH3 foci observed among the analyzed ‘n’ number of embryos. Last column: the haploid percent and the number of plants assays is indicated. Note: 5 smaller-sized CENH3 signals are indicated with an arrow in row E. Haploid frequency of (C) is determined by flow cytometry of nuclei isolated from seeds, whereas in the case of (D-G), gl1-1 plants were used as a proxy for CENH3(+/+) and haploid frequency was determined by glabrous offsprings.\u003c/p\u003e","description":"","filename":"Fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-5974840/v1/4cad8e807c56591c2426169b.png"},{"id":76075448,"identity":"1ada888b-89aa-4995-aff7-25ec0ccea9f7","added_by":"auto","created_at":"2025-02-12 05:32:50","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":29690,"visible":true,"origin":"","legend":"\u003cp\u003eEgg cell-specific degradation of EYFP-CENH3 results in shriveled seeds and increased haploid induction frequency. A) Schematic representation of different constructs (EV-SPOP, EV-NSlmb, EV) used for the egg cell-specific degradation of EYFP-CENH3. PPT- Phosphinothricin B) Frequency of phosphinothricin-resistant seedlings obtained from T2 seeds of 10 individual T1 [cenh3(-/-), EYFP-gCENH3(+/+)] plants for the different constructs shown in (A). Comparisons are based upon adjusted p values based on Holm-sidak method for three pairwise comparisons C) Frequency of shriveled seeds after self-pollination from 10 different T1 plants from each genotypes ([cenh3(-/-), EYFP-gCENH3(+/+)] and [CENH3(+/+), EYFP-gCENH3(+/+)]) transformed with the different constructs shown in (A). p values represent a two-sample t-test. D) Haploidization frequency of independent [cenh3(-/-), EYFP-gCENH3(+/+)] T1 plants hemizygous for the different constructs shown in (A), after pollination with pollen from gl1-1plants. [cenh3(-/-), EYFP-gCENH3(+/+)] plants without any of the constructs were used as control. ‘n’ indicates the individual T1 plants evaluated for each construct and individual plants used as of control. ‘N’ indicates total number of plants screened for the glabrous phenotype from all the T1 plants evaluated. p values are adjusted p values based on Holm-sidak method for six pairwise comparisons.\u003c/p\u003e","description":"","filename":"Fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-5974840/v1/dd7b81f25cac517a6d79c5d1.png"},{"id":76075449,"identity":"ab24d75c-00e2-42ae-9396-879ed480ec41","added_by":"auto","created_at":"2025-02-12 05:32:50","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":30237,"visible":true,"origin":"","legend":"\u003cp\u003eEgg cell-specific degradation of ALFA-CENH3 by different E3-ligase results in haploid induction. A) Schematic representation of different constructs (EN-SPOP, EN-NSlmb, EN \u0026amp; Control) used for the egg cell-specific degradation of ALFA-CENH3. PPT – Phosphinothricin; FAST-red – Fluorescent seed marker; Barnase – expressed under the control of A9 promoter for male sterility. B) Haploidization frequency of independent [cenh3(-/-), ALFA-gCENH3(+/+)] T1 plants hemizygous for the different constructs shown in (A), after pollination with pollen from gl1-1 plants. C) Schematic representation of different constructs harboring plant-derived E3-ligases (EN-EL5, EN-BPM1 \u0026amp; Control) used for the egg cell-specific degradation of ALFA-CENH3. D) Haploidization frequency of independent [cenh3(-/-), ALFA-gCENH3(+/+)] T1 plants hemizygous for the different constructs shown in (C), after pollination with pollen from gl1-1 plants. B \u0026amp; D) ‘n’ indicates the individual T1 plants evaluated for each construct. ‘N’ indicates total number of plants screened for the glabrous phenotype from all the T1 plants evaluated. p values in B) is based upon a two-sample t-test whereas in D) is based upon the Mann-Whitney rank sum test\u003c/p\u003e","description":"","filename":"Fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-5974840/v1/cbba6f77f743ee354ec39f31.png"},{"id":79147530,"identity":"427d5d98-cb27-45a2-9670-24f05f384917","added_by":"auto","created_at":"2025-03-25 03:23:49","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":668145,"visible":true,"origin":"","legend":"Article File","description":"","filename":"Manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5974840/v1_covered_2f985d5f-ecec-47d3-8061-7d783a5f1114.pdf"},{"id":76075445,"identity":"1b32d7db-110b-41b1-bb1d-ff240825b77f","added_by":"auto","created_at":"2025-02-12 05:32:50","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":568195,"visible":true,"origin":"","legend":"","description":"","filename":"ExtendedFigures.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5974840/v1/f0adcb24c6ff267514ca8877.pdf"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Gametophytic degradation of CENH3 - a synthetic biology approach for haploid induction in plants","fulltext":[],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":false,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":true,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"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":"","lastPublishedDoi":"10.21203/rs.3.rs-5974840/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5974840/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Double haploid technology is a critical tool for accelerating plant breeding. The manipulation of centromere-specific histone H3 (CENH3) has previously been shown to induce haploid progeny in both the model plant Arabidopsis and certain crop species. However, a universally applicable strategy for CENH3-based haploid induction across diverse crop species is still lacking. Here, we demonstrate that extreme asymmetry in centromeric CENH3 levels between parental chromosomes is a causal factor for eliminating chromosomes with ‘weaker’ centromeres during early embryogenesis. Consequently, engineered E3-ligases were utilized to selectively degrade CENH3 in the egg cell to induce wild-type paternal haploids. Also, we attempted to optimize the CENH3 degradation using E3-ligases of plant-origin. Our findings provide valuable insights to enhance the haploid induction efficiency further. 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