Knocking-out the brassinosteroid biosynthesis genes DWARF1 or DWARF4 produces upright canopy architecture in wheat

Knocking-out the brassinosteroid biosynthesis genes DWARF1 or DWARF4 produces upright canopy architecture in wheat | 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 Knocking-out the brassinosteroid biosynthesis genes DWARF1 or DWARF4 produces upright canopy architecture in wheat Manpartik S. Gill, Andrew L. Phillips, Danuše Tarkowská, John Addy, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6982644/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: Brassinosteroids (BR) regulate multiple agronomic traits in plants, including leaf angle. BR depletion promotes an upright canopy architecture, which can improve vertical light distribution and grain yields in cereals under high planting densities. However, broad reductions in BR activity often have negative impacts on fertility and grain size. Spatially restricted modification of BR metabolism through targeting specific genes could, therefore, provide an erect canopy while minimising negative pleiotropic effects. In the BR biosynthesis pathway, DWARF1/DIMINUTO ( DWF1/DIM ) encodes a C-24 reductase that catalyses the production of the first committed sterol BR precursor, while DWF4 and DWF11 encode 22α-hydroxylase enzymes that act later in the pathway, and which exhibit partial functional redundancy. Results: Using ethyl-methanesulfonate (EMS)-induced mutations that introduce premature stop codons, we generated wheat lines lacking functional copies of all three homoeologues of DWF1 and DWF4 in the Cadenza background. The dwf4-abd mutant displayed upright leaves and a slight height reduction but showed no differences in grain size or weight when tested in glasshouse and field conditions. The dwf1-abd mutant also produced an upright canopy, but tiller number, grain size and grain weight were reduced compared to the wild-type. Scanning electron microscopy revealed that the reduced leaf angle in these mutants was due to reduced cell elongation in the auricle region of the lamina joint, causing the leaf blade to be more tightly packed around the ligule. The dwf1-abd mutant was hypersensitive to exogenous BR application. Metabolite profiling showed that concentrations of the BR precursors campesterol (CR), campestanol (CN) and 6-oxoCN were significantly reduced in dwf1-abd seedlings compared to wild-type controls, whereas levels of the bioactive BRs castasterone (CS) and brassinolide (BL) were unexpectedly elevated in both dwf1-abd and dwf4-abd . Conclusion: Wheat dwf4 null mutations confer an erect canopy architecture without yield penalties, suggesting this gene may be a promising target to develop ideotypes suitable for high-density planting. By contrast, dwf1 null mutants exhibit negative pleiotropic effects and are unlikely to be useful for agriculture. The characterised mutants advance our understanding of BR metabolism and can be explored for their potential to improve grain yields. wheat brassinosteroids leaf angle ligule plant height grain size Full Text Additional Declarations No competing interests reported. Supplementary Files Supplementaryfiles.docx Figure S1: DWF1 and DWF4 homoeologues and mutant alleles used in this study. Figure S2: Phenotypic characterization of all combinatorial dwf1 and dwf4 mutants under glasshouse conditions. Figure S3: Phenotype of Cadenza and dwf1-abd mutant at the tillering stage in the field. Figure S4: High throughput phenotyping of dwf1-abd mutant under Field Scanalyzer. Figure S5: Expression of DWF1 genes in wheat variety ‘Azhurnaya’. Figure S6: Expression of DWF4 and DWF11 genes in wheat variety ‘Azhurnaya’. Table S1: Homoeologue-specific primers designed to amplify fragments around the deleterious mutations in TaDWF1 and TaDWF4 genes. Table S2: KASP primers designed to differentiate the mutant and wild type allele in segregating DWF1 and DWF4 populations. Table S3: BR levels (pg/mg DW) in dwf1-abd and dwf4-abd mutants and controls. 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-6982644","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":483544495,"identity":"a14e35aa-8b34-4c13-ab70-e8ad7af9140d","order_by":0,"name":"Manpartik S. Gill","email":"","orcid":"","institution":"Rothamsted Research","correspondingAuthor":false,"prefix":"","firstName":"Manpartik","middleName":"S.","lastName":"Gill","suffix":""},{"id":483544496,"identity":"ea779b99-d49a-4103-beea-5f4e9ec0ba61","order_by":1,"name":"Andrew L. 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17:21:17","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":2929278,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFigure S1: \u003c/strong\u003e\u003cem\u003eDWF1 \u003c/em\u003eand\u003cem\u003e DWF4\u003c/em\u003ehomoeologues and mutant alleles used in this study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFigure S2: \u003c/strong\u003ePhenotypic characterization of all combinatorial \u003cem\u003edwf1\u003c/em\u003e and \u003cem\u003edwf4\u003c/em\u003e mutants under glasshouse conditions.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFigure S3:\u003c/strong\u003e Phenotype of Cadenza and \u003cem\u003edwf1-abd\u003c/em\u003emutant at the tillering stage in the field.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFigure S4: \u003c/strong\u003eHigh throughput phenotyping of \u003cem\u003edwf1-abd\u003c/em\u003emutant under Field Scanalyzer.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFigure S5:\u003c/strong\u003e Expression of \u003cem\u003eDWF1\u003c/em\u003e genes in wheat variety ‘Azhurnaya’.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFigure S6: \u003c/strong\u003eExpression of \u003cem\u003eDWF4\u003c/em\u003e and \u003cem\u003eDWF11\u003c/em\u003egenes in wheat variety ‘Azhurnaya’.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable S1: \u003c/strong\u003eHomoeologue-specific primers designed to amplify fragments around the deleterious mutations in \u003cem\u003eTaDWF1 \u003c/em\u003eand\u003cem\u003e TaDWF4 \u003c/em\u003egenes.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable S2: \u003c/strong\u003eKASP primers designed to differentiate the mutant and wild type allele in segregating \u003cem\u003eDWF1 \u003c/em\u003eand\u003cem\u003e DWF4 \u003c/em\u003epopulations.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable S3: \u003c/strong\u003eBR levels (pg/mg DW) in \u003cem\u003edwf1-abd \u003c/em\u003eand\u003cem\u003edwf4-abd\u003c/em\u003e mutants and controls.\u003c/p\u003e","description":"","filename":"Supplementaryfiles.docx","url":"https://assets-eu.researchsquare.com/files/rs-6982644/v1/0835856d30ea397e02fe44ef.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Knocking-out the brassinosteroid biosynthesis genes DWARF1 or DWARF4 produces upright canopy architecture in wheat","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":"wheat, brassinosteroids, leaf angle, ligule, plant height, grain size","lastPublishedDoi":"10.21203/rs.3.rs-6982644/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6982644/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground: \u003c/strong\u003eBrassinosteroids (BR) regulate multiple agronomic traits in plants, including leaf angle. BR depletion promotes an upright canopy architecture, which can improve vertical light distribution and grain yields in cereals under high planting densities. \u0026nbsp;However, broad reductions in BR activity often have negative impacts on fertility and grain size. Spatially restricted modification of BR metabolism through targeting specific genes could, therefore, provide an erect canopy while minimising negative pleiotropic effects. In the BR biosynthesis pathway, \u003cem\u003eDWARF1/DIMINUTO \u003c/em\u003e(\u003cem\u003eDWF1/DIM\u003c/em\u003e) encodes a C-24 reductase that catalyses the production of the first committed sterol BR precursor, while \u003cem\u003eDWF4 \u003c/em\u003eand \u003cem\u003eDWF11 \u003c/em\u003eencode 22α-hydroxylase enzymes that act later in the pathway, and which exhibit partial functional redundancy.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults: \u003c/strong\u003eUsing ethyl-methanesulfonate (EMS)-induced mutations that introduce premature stop codons, we generated wheat lines lacking functional copies of all three homoeologues of \u003cem\u003eDWF1\u003c/em\u003e and \u003cem\u003eDWF4 \u003c/em\u003ein the Cadenza background. The \u003cem\u003edwf4-abd\u003c/em\u003emutant displayed upright leaves and a slight height reduction but showed no differences in grain size or weight when tested in glasshouse and field conditions. The \u003cem\u003edwf1-abd\u003c/em\u003e mutant also produced an upright canopy, but tiller number, grain size and grain weight were reduced compared to the wild-type.\u003c/p\u003e\n\u003cp\u003eScanning electron microscopy revealed that the reduced leaf angle in these mutants was due to reduced cell elongation in the auricle region of the lamina joint, causing the leaf blade to be more tightly packed around the ligule. The \u003cem\u003edwf1-abd\u003c/em\u003emutant was hypersensitive to exogenous BR application. Metabolite profiling showed that concentrations of the BR precursors campesterol (CR), campestanol (CN) and 6-oxoCN were significantly reduced in \u003cem\u003edwf1-abd \u003c/em\u003eseedlings compared to wild-type controls, whereas levels of the bioactive BRs castasterone (CS) and brassinolide (BL) were unexpectedly elevated in both \u003cem\u003edwf1-abd \u003c/em\u003eand \u003cem\u003edwf4-abd\u003c/em\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion: \u003c/strong\u003eWheat \u003cem\u003edwf4 \u003c/em\u003enull mutations confer an erect canopy architecture without yield penalties, suggesting this gene may be a promising target to develop ideotypes suitable for high-density planting. By contrast, \u003cem\u003edwf1 \u003c/em\u003enull mutants exhibit negative pleiotropic effects and are unlikely to be useful for agriculture. The characterised mutants advance our understanding of BR metabolism and can be explored for their potential to improve grain yields.\u003c/p\u003e","manuscriptTitle":"Knocking-out the brassinosteroid biosynthesis genes DWARF1 or DWARF4 produces upright canopy architecture in wheat","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-14 17:05:12","doi":"10.21203/rs.3.rs-6982644/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":"32e9d9a1-f818-4a93-894a-6c855ebf85b8","owner":[],"postedDate":"July 14th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-02-11T12:26:31+00:00","versionOfRecord":[],"versionCreatedAt":"2025-07-14 17:05:12","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6982644","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6982644","identity":"rs-6982644","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}