Genetic foundations of copper stress tolerance in safflower: bridging heavy metal contamination and global warming challenges

Genetic foundations of copper stress tolerance in safflower: bridging heavy metal contamination and global warming challenges | 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 Genetic foundations of copper stress tolerance in safflower: bridging heavy metal contamination and global warming challenges Fawad Ali, Arif Ali, Obaid Ullah Shah, Mian Arif, Muhammad Azhar Nadeem, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9539129/v1 This work is licensed under a CC BY 4.0 License Status: Under Revision Version 1 posted 14 You are reading this latest preprint version Abstract Copper (Cu) contamination threatens agriculture, yet safflower's genetic basis for Cu tolerance remains unexplored—especially as global warming intensifies risks through nighttime warming and soil acidification. We conducted a pot experiment using 94 safflower genotypes under four copper chloride (CuCl₂) concentrations, revealing substantial variation among genotypes and treatments with trait reductions ranging from 1.75% (root-shoot ratio) (RSR) to 57.14% (dry root weight) (DRW). Correlation analysis showed positive trait relationships, while principal component analysis (PCA) grouped genotypes into three clusters. Ten genotypes consistently performed better under Cu stress, indicating strong tolerance potential for climate-resilient breeding. Genome-wide association study (GWAS) detected 180 significant marker-trait associations (MTAs) across eight traits, and gene annotation linked candidate genes to brassinosteroid signaling, ion homeostasis, metal response, and transport. Co-expression analysis revealed a tightly connected tolerance module in which AT5G01610 and AT5G14640 shared the same top co-expressed partners ( r > 0.8), including HMA4 , CAX1 , and SOS1 , suggesting coordinated roles in Cu stress tolerance. Convergent protein-protein interaction (PPI) validation confirmed these candidates as distinct hubs that are physically interacting and transcriptionally co-regulated, supporting their function as Cu tolerance modules. In silico heatmap-based expression profiling showed distinct, stress-specific transcriptional responses in Arabidopsis roots, with Cu inducing, cadmium (Cd) eliciting mixed, and (Al) aluminum repressing expression. RNA-seq analysis identified seven Cu stress tolerance genes, validated by qRT-PCR. These findings highlight promising genotypes for developing Cu-resilient safflower cultivars, while further investigation of the highly expressed genes is needed to clarify their roles under Cu stress. Genome-wide association study Stress tolerance index Candidate genes Expression visualization qRT-PCR Full Text Additional Declarations No competing interests reported. Supplementary Files FigureS2.png FigureS3.png tableS1.docx TableS2.docx TableS3.docx TableS4.docx Cite Share Download PDF Status: Under Revision Version 1 posted Editorial decision: Revision requested 14 May, 2026 Reviewers agreed at journal 11 May, 2026 Reviews received at journal 11 May, 2026 Reviewers agreed at journal 08 May, 2026 Reviews received at journal 07 May, 2026 Reviewers agreed at journal 07 May, 2026 Reviewers agreed at journal 07 May, 2026 Reviewers agreed at journal 07 May, 2026 Reviewers agreed at journal 06 May, 2026 Reviewers agreed at journal 06 May, 2026 Reviewers invited by journal 06 May, 2026 Editor assigned by journal 02 May, 2026 Submission checks completed at journal 02 May, 2026 First submitted to journal 27 Apr, 2026 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-9539129","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":638616758,"identity":"05fd1466-cdd0-4a69-8b85-02cd58e7806d","order_by":0,"name":"Fawad Ali","email":"","orcid":"","institution":"Jeju National University","correspondingAuthor":false,"prefix":"","firstName":"Fawad","middleName":"","lastName":"Ali","suffix":""},{"id":638616759,"identity":"e777b162-4892-4692-9a6f-08d611d4f129","order_by":1,"name":"Arif Ali","email":"","orcid":"","institution":"Hainan University","correspondingAuthor":false,"prefix":"","firstName":"Arif","middleName":"","lastName":"Ali","suffix":""},{"id":638616760,"identity":"823074c9-9e08-4565-8cc4-d38ef227fe0f","order_by":2,"name":"Obaid Ullah Shah","email":"","orcid":"","institution":"Shanghai Jiao Tong University","correspondingAuthor":false,"prefix":"","firstName":"Obaid","middleName":"Ullah","lastName":"Shah","suffix":""},{"id":638616761,"identity":"08810310-e42a-40de-9dee-5ba6bfdbb31c","order_by":3,"name":"Mian Arif","email":"","orcid":"","institution":"Nuclear Institute for Agriculture and Biology","correspondingAuthor":false,"prefix":"","firstName":"Mian","middleName":"","lastName":"Arif","suffix":""},{"id":638616762,"identity":"3e668e9a-10ee-4f5d-9b06-bf6776a01735","order_by":4,"name":"Muhammad Azhar Nadeem","email":"","orcid":"","institution":"Mersin University","correspondingAuthor":false,"prefix":"","firstName":"Muhammad","middleName":"Azhar","lastName":"Nadeem","suffix":""},{"id":638616763,"identity":"bd08306c-5962-4b62-a741-366945c5d742","order_by":5,"name":"Faheem Shehzad Baloch","email":"","orcid":"","institution":"Mersin University","correspondingAuthor":false,"prefix":"","firstName":"Faheem","middleName":"Shehzad","lastName":"Baloch","suffix":""},{"id":638616764,"identity":"7ca9219d-c09d-4a48-8ece-1b03338a5817","order_by":6,"name":"Syed Mansoor Jan","email":"","orcid":"","institution":"Shanghai Jiao Tong University","correspondingAuthor":false,"prefix":"","firstName":"Syed","middleName":"Mansoor","lastName":"Jan","suffix":""},{"id":638616765,"identity":"88b86a2d-7685-453d-9874-66638a2c287d","order_by":7,"name":"My Khanh Thi Ha","email":"","orcid":"","institution":"Duy Tan University","correspondingAuthor":false,"prefix":"","firstName":"My","middleName":"Khanh Thi","lastName":"Ha","suffix":""},{"id":638616766,"identity":"c45af3c3-d0b8-4199-b6dc-e7e5fc380721","order_by":8,"name":"Yong Suk Chung","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA5klEQVRIiWNgGAWjYHACNhAhx9jAkIAsQFiLMelaEhvQBHADg/OHnz3m3VGb3tze8Ey6sI1Bnr+BLe0DXi030syNec8cz23sOZAmPbONwXDGAbbDM/Br4WGT5m07lts4IyENyGBg3MDA3kzAYWfAWtIZ5z8Aa7EnrOVADkhLTQLjDAawlsQNDGyH8WqRvJFmJjm37YBhY09CsjXPOYnkGYfZkvFq4QOGmMTbtjp5w/Yzibd5ymxs+9vbjPFqUTgApg4zGDbwJAAZEgwMzHg1MDDIN4CpOgZ5BvYDBNSOglEwCkbBSAUAl+dE+EBc7xAAAAAASUVORK5CYII=","orcid":"","institution":"Jeju National University","correspondingAuthor":true,"prefix":"","firstName":"Yong","middleName":"Suk","lastName":"Chung","suffix":""}],"badges":[],"createdAt":"2026-04-27 09:08:48","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9539129/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9539129/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":109312120,"identity":"6ac8ba35-88cd-48a6-8683-e5511a3f90b6","added_by":"auto","created_at":"2026-05-15 11:32:08","extension":"png","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":523528,"visible":true,"origin":"","legend":"","description":"","filename":"FigureS2.png","url":"https://assets-eu.researchsquare.com/files/rs-9539129/v1/9ae2115f6826bcae3e5e79df.png"},{"id":109405426,"identity":"dab9350b-632c-42cb-9c84-b907812080c7","added_by":"auto","created_at":"2026-05-17 13:17:58","extension":"png","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":672247,"visible":true,"origin":"","legend":"","description":"","filename":"FigureS3.png","url":"https://assets-eu.researchsquare.com/files/rs-9539129/v1/fdf8c692b922bf830df70a8d.png"},{"id":109405462,"identity":"8e49e936-1b52-42b5-9bc8-dcd254e3d608","added_by":"auto","created_at":"2026-05-17 13:18:19","extension":"docx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":20738,"visible":true,"origin":"","legend":"","description":"","filename":"tableS1.docx","url":"https://assets-eu.researchsquare.com/files/rs-9539129/v1/ea265f132358cadd595e1418.docx"},{"id":109312123,"identity":"1f536f29-dfa9-4d09-9a6b-4698b86c7f11","added_by":"auto","created_at":"2026-05-15 11:32:08","extension":"docx","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":15764,"visible":true,"origin":"","legend":"","description":"","filename":"TableS2.docx","url":"https://assets-eu.researchsquare.com/files/rs-9539129/v1/475e1afdec958bec1a4d4611.docx"},{"id":109312124,"identity":"e197e68e-042c-4b16-bc21-cc44a1038e9c","added_by":"auto","created_at":"2026-05-15 11:32:08","extension":"docx","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":37860,"visible":true,"origin":"","legend":"","description":"","filename":"TableS3.docx","url":"https://assets-eu.researchsquare.com/files/rs-9539129/v1/cb1470166f5e044009554e87.docx"},{"id":109312126,"identity":"85ef55aa-9330-4962-9956-8fcf2348e1fb","added_by":"auto","created_at":"2026-05-15 11:32:08","extension":"docx","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":17845,"visible":true,"origin":"","legend":"","description":"","filename":"TableS4.docx","url":"https://assets-eu.researchsquare.com/files/rs-9539129/v1/1f44ef15ee8985f4bc2044b7.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Genetic foundations of copper stress tolerance in safflower: bridging heavy metal contamination and global warming challenges","fulltext":[],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":false,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":true,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":true,"isPdfUpToDate":false,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"plant-cell-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pcre","sideBox":"Learn more about [Plant Cell Reports](https://www.springer.com/journal/299)","snPcode":"299","submissionUrl":"https://submission.nature.com/new-submission/299/3","title":"Plant Cell Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Genome-wide association study, Stress tolerance index, Candidate genes, Expression visualization, qRT-PCR","lastPublishedDoi":"10.21203/rs.3.rs-9539129/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9539129/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eCopper (Cu) contamination threatens agriculture, yet safflower's genetic basis for Cu tolerance remains unexplored—especially as global warming intensifies risks through nighttime warming and soil acidification. We conducted a pot experiment using 94 safflower genotypes under four copper chloride (CuCl₂) concentrations, revealing substantial variation among genotypes and treatments with trait reductions ranging from 1.75% (root-shoot ratio) (RSR) to 57.14% (dry root weight) (DRW). Correlation analysis showed positive trait relationships, while principal component analysis (PCA) grouped genotypes into three clusters. Ten genotypes consistently performed better under Cu stress, indicating strong tolerance potential for climate-resilient breeding. Genome-wide association study (GWAS) detected 180 significant marker-trait associations (MTAs) across eight traits, and gene annotation linked candidate genes to brassinosteroid signaling, ion homeostasis, metal response, and transport. Co-expression analysis revealed a tightly connected tolerance module in which \u003cem\u003eAT5G01610\u003c/em\u003e and \u003cem\u003eAT5G14640\u003c/em\u003eshared the same top co-expressed partners (\u003cem\u003er\u003c/em\u003e \u0026gt; 0.8), including \u003cem\u003eHMA4\u003c/em\u003e, \u003cem\u003eCAX1\u003c/em\u003e, and \u003cem\u003eSOS1\u003c/em\u003e, suggesting coordinated roles in Cu stress tolerance. Convergent protein-protein interaction (PPI) validation confirmed these candidates as distinct hubs that are physically interacting and transcriptionally co-regulated, supporting their function as Cu tolerance modules. \u003cem\u003eIn silico\u003c/em\u003e heatmap-based expression profiling showed distinct, stress-specific transcriptional responses in \u003cem\u003eArabidopsis\u003c/em\u003e roots, with Cu inducing, cadmium (Cd) eliciting mixed, and (Al) aluminum repressing expression. RNA-seq analysis identified seven Cu stress tolerance genes, validated by qRT-PCR. These findings highlight promising genotypes for developing Cu-resilient safflower cultivars, while further investigation of the highly expressed genes is needed to clarify their roles under Cu stress.\u003c/p\u003e","manuscriptTitle":"Genetic foundations of copper stress tolerance in safflower: bridging heavy metal contamination and global warming challenges","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-05-15 11:32:04","doi":"10.21203/rs.3.rs-9539129/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-05-15T03:50:51+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"245180028366825535010297133742081646881","date":"2026-05-12T01:16:09+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-11T06:33:09+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"209998747827682872085940295497590095457","date":"2026-05-08T09:49:28+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-07T15:45:26+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"36434448997461519681241773790342240285","date":"2026-05-07T14:30:21+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"70253180250267934596821237078031045092","date":"2026-05-07T10:52:41+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"88220630182635946247596248288540663892","date":"2026-05-07T07:28:37+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"275667955302763775637254697178598973214","date":"2026-05-06T19:57:46+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"146601535973351550770502699859350122770","date":"2026-05-06T11:22:58+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-05-06T11:15:34+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-05-02T08:23:24+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-05-02T07:25:32+00:00","index":"","fulltext":""},{"type":"submitted","content":"Plant Cell Reports","date":"2026-04-27T08:54:19+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"plant-cell-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pcre","sideBox":"Learn more about [Plant Cell Reports](https://www.springer.com/journal/299)","snPcode":"299","submissionUrl":"https://submission.nature.com/new-submission/299/3","title":"Plant Cell Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"f861b451-7e4b-4662-b303-e6eda4b05956","owner":[],"postedDate":"May 15th, 2026","published":true,"recentEditorialEvents":[{"type":"decision","content":"Revision requested","date":"2026-05-15T03:50:51+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"245180028366825535010297133742081646881","date":"2026-05-12T01:16:09+00:00","index":40,"fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-11T06:33:09+00:00","index":39,"fulltext":""},{"type":"reviewerAgreed","content":"209998747827682872085940295497590095457","date":"2026-05-08T09:49:28+00:00","index":36,"fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-07T15:45:26+00:00","index":35,"fulltext":""},{"type":"reviewerAgreed","content":"36434448997461519681241773790342240285","date":"2026-05-07T14:30:21+00:00","index":34,"fulltext":""},{"type":"reviewerAgreed","content":"70253180250267934596821237078031045092","date":"2026-05-07T10:52:41+00:00","index":33,"fulltext":""},{"type":"reviewerAgreed","content":"88220630182635946247596248288540663892","date":"2026-05-07T07:28:37+00:00","index":31,"fulltext":""},{"type":"reviewerAgreed","content":"275667955302763775637254697178598973214","date":"2026-05-06T19:57:46+00:00","index":30,"fulltext":""},{"type":"reviewerAgreed","content":"146601535973351550770502699859350122770","date":"2026-05-06T11:22:58+00:00","index":26,"fulltext":""},{"type":"reviewersInvited","content":"22","date":"2026-05-06T11:15:34+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-05-02T08:23:24+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-05-02T07:25:32+00:00","index":"","fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"in-revision","subjectAreas":[],"tags":[],"updatedAt":"2026-05-15T11:32:04+00:00","versionOfRecord":[],"versionCreatedAt":"2026-05-15 11:32:04","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9539129","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9539129","identity":"rs-9539129","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}