Integrative transcriptomic and HPTL-ESI-MS analysis reveals a strong coordination between genes and pathways contributing to high erucic triacylglycerol biosynthesis during Pennycress (Thlaspi arvense L.) seed maturation

Integrative transcriptomic and HPTL-ESI-MS analysis reveals a strong coordination between genes and pathways contributing to high erucic triacylglycerol biosynthesis during Pennycress (Thlaspi arvense L.) seed maturation | 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 Integrative transcriptomic and HPTL-ESI-MS analysis reveals a strong coordination between genes and pathways contributing to high erucic triacylglycerol biosynthesis during Pennycress (Thlaspi arvense L.) seed maturation Ana Claver, María Ángeles Luján, José Manuel Escuín, María Savirón, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3221172/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 Thlaspi arvense (Pennycress) is an emerging feedstock for biofuel production because of its high seed-oil content enriched in erucic acid (35-40% of total fatty acids). A transcriptomic analysis together with an HPTLC-ESI-MS and MS n positional analysis characterization of the TAG fractions in five different seed maturation stages was performed to study the high erucic seed-oil biosynthesis in Pennycress. The RNA-Seq data showed a temporal pattern of regulation in which genes involved in the synthesis of carbon precursors and fatty acids were highly abundant at the earlier stages of maturation, then decreasing in the mature ones. Genes involved in TAG biosynthesis showed a complex temporal regulation in which DGAT2 and PDAT1 showed higher expression at the earlier stages of maturation, coincident with the rapid incorporation of 22:1 to TAG. Genes from the acyl-editing pathway showed higher expression at the earlier stages of seed maturation, suggesting a major role of this pathway at the initial stages of seed-oil biosynthesis. HPTLC-ESI-MS analysis showed an increase of TAG species containing VLCFAs (22:1 and 24:1) with 18:2 at sn-2 position with seed maturation. This increase was concomitant with an increase of DGAT1 and PDAT2 mRNA levels, suggesting a higher Kennedy pathway activity. However, the concerted regulation between DGAT and PDAT enzymes suggested a coordination between pathways, isoforms and substrate pools for the synthesis of high erucic TAG in Pennycress. Thlaspi arvense seed oil TAG DGAT PDAT VLCFAs erucic acid. Full Text Supplementary Files Fig.S1.docx Fig. S1. Differential expression of genes encoding transcription factors. (a) Distribution of DEGs among different transcription factor families. (b) Differential expression of genes encoding transcription factors for each of the five seed maturation stages used in this work. Values represent average log2(fpkm +1) values from each of the biological repeats and were used to generate heatmaps from http://bar.toronto.ca/ntools/cgi- 41 bin/ntools_heatmapper_plus.cgi. The Thlaspi arvense annotated genome (Nunn et al., 2022) was used for identification of the gene ID. Fig.S2.docx Fig. S2. Expression profiling of individual genes and isoforms involved in oil bodies formation during seed maturation by RNA-Seq (light grey bars) and qPCR (dark grey bars). For RNA-Seq data, expression levels represented by FPKM values were normalized to GREEN stage. The genes analysed, OLE1, OLE2, and OBA1a, are indicated in each figure. For qPCR analysis, data were obtained from three independent pools of seeds from five plants of each line. Data represent means of at least three biological replicates. Fig.S3.docx Fig. S3. HPTLC chromatograms of standards at UV 190 nm: pic1: 1-Oleoyl-rac-glycerol, pic 2: 1,2-dioleoyl-rac-glycerol, pic 3: linoleic acid, pic 4: oleic acid, pic 5: erucic acid, pic 6: glyceryl trioleate, pic 7: methyl oleate, pic 8: cholesteryl stearate, and pic 9: cholesteryl oleate. Fig.S4.docx Fig. S4. HPTLC chromatograms corresponding to samples at the different maturation stages detected at UV 190 nm: a) GREEN, b) GREENYELLOW, c) YELLOWGREEN; d) YELLOW, and e) MATURE. Fig.S5.docx Fig. S5. HPTLC-ESI+ -MS/MS of molecular species found in separated and extracted TAG peak (907.8, 935.9, 959.9, 989.9 and 1046.0) tableS1primers.docx Table S1 primers used for qPCR analysis in this study. TableS2.QCdatastatistics.xlsx Table S2. Total reads, clean reads and quality parameters of the RNA-Seq data. SeedmaturationstagesRNAseqdeg.xlsx Supporting information 1: RNA-Seq data set containing all the results for each pairwise comparison used in this study. 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-3221172","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":223191834,"identity":"08bc643e-3ffa-4629-9a79-c89302f725f9","order_by":0,"name":"Ana Claver","email":"","orcid":"","institution":"EEAD - CSIC Zaragoza: Estacion Experimental de Aula Dei","correspondingAuthor":false,"prefix":"","firstName":"Ana","middleName":"","lastName":"Claver","suffix":""},{"id":223191835,"identity":"deb9d7d0-1b3d-40b4-afd3-8e03ffbf4a6a","order_by":1,"name":"María Ángeles Luján","email":"","orcid":"","institution":"EEAD - CSIC Zaragoza: Estacion Experimental de Aula Dei","correspondingAuthor":false,"prefix":"","firstName":"María","middleName":"Ángeles","lastName":"Luján","suffix":""},{"id":223191836,"identity":"ca7d17d5-8665-457f-84db-cdfabadb6130","order_by":2,"name":"José Manuel Escuín","email":"","orcid":"","institution":"Instituto de Carboquímica: Instituto de Carboquimica","correspondingAuthor":false,"prefix":"","firstName":"José","middleName":"Manuel","lastName":"Escuín","suffix":""},{"id":223191837,"identity":"593b9cde-1286-4884-a57c-c93908cf70b8","order_by":3,"name":"María Savirón","email":"","orcid":"","institution":"University of Zaragoza: Universidad de Zaragoza","correspondingAuthor":false,"prefix":"","firstName":"María","middleName":"","lastName":"Savirón","suffix":""},{"id":223191838,"identity":"deed21d8-180a-4bae-9aac-2a8895679656","order_by":4,"name":"María Victoria López","email":"","orcid":"","institution":"EEAD - CSIC Zaragoza: Estacion Experimental de Aula Dei","correspondingAuthor":false,"prefix":"","firstName":"María","middleName":"Victoria","lastName":"López","suffix":""},{"id":223191839,"identity":"e0a5b009-2ebc-46f7-831e-e0283834fc3f","order_by":5,"name":"Rafael Picorel","email":"","orcid":"","institution":"EEAD - CSIC Zaragoza: Estacion Experimental de Aula Dei","correspondingAuthor":false,"prefix":"","firstName":"Rafael","middleName":"","lastName":"Picorel","suffix":""},{"id":223191840,"identity":"031b49cd-360f-47e7-9e9f-a655eb271989","order_by":6,"name":"Carmen Jarne","email":"","orcid":"","institution":"Universidad de Zaragoza","correspondingAuthor":false,"prefix":"","firstName":"Carmen","middleName":"","lastName":"Jarne","suffix":""},{"id":223191841,"identity":"2d59950d-32d8-4f8b-8a87-9691d3399960","order_by":7,"name":"Vicente Cebolla","email":"","orcid":"","institution":"ICB: Instituto de Carboquimica","correspondingAuthor":false,"prefix":"","firstName":"Vicente","middleName":"","lastName":"Cebolla","suffix":""},{"id":223191842,"identity":"ef0593a9-9377-4061-8462-6225e73eca5d","order_by":8,"name":"Miguel Alfonso","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA3klEQVRIiWNgGAWjYDACdsYHIEoOREgAMQ9hLczMBiDKmHQtiQ1QLYQBfzMz4+OCPzbp/ezND28w1NTJ8DcwH/6AT4vEYWZm45ltabkze44ZWzAcO8wjcYAtDb91h/mPSfM2HM7dcCPBTIKx4QCPAQOPGV4d8oeZ2aR5/hxOt7///BtQSx1QC/9nvA4zAGthO5xgIMEDsoUZZAv+cDAE+YW3Lc1wxpmcYosEkF8Os5nh1SJ3vJnxMc8fG3n+9uMbb3yoqbPnb29+jNdhqCABRDATr34UjIJRMApGAQ4AALosPZvmX0IMAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0002-8190-5156","institution":"Estacion Experimental de Aula Dei","correspondingAuthor":true,"prefix":"","firstName":"Miguel","middleName":"","lastName":"Alfonso","suffix":""}],"badges":[],"createdAt":"2023-07-31 14:52:39","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3221172/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3221172/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":43243667,"identity":"f22045d0-bf15-4257-abe4-f3d649a8dc6f","added_by":"auto","created_at":"2023-09-16 19:53:38","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":6440457,"visible":true,"origin":"","legend":"","description":"","filename":"ClaveretalTAGpennycressFINAL.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3221172/v1_covered_c63ae07f-2bb1-4b87-b7a7-1c39a17fa786.pdf"},{"id":41158026,"identity":"c8e82749-35ba-42f5-a8fb-902cfc6a653b","added_by":"auto","created_at":"2023-08-07 06:53:54","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":1561350,"visible":true,"origin":"","legend":"\u003cp\u003eFig. S1. Differential expression of genes encoding transcription factors. (a) Distribution \u0026nbsp;of DEGs among different transcription factor families. (b) Differential expression of \u0026nbsp;genes encoding transcription factors for each of the five seed maturation stages used in \u0026nbsp;this work. Values represent average log2(fpkm +1) values from each of the biological \u0026nbsp;repeats and were used to generate heatmaps from http://bar.toronto.ca/ntools/cgi- 41 \u0026nbsp;bin/ntools_heatmapper_plus.cgi. The Thlaspi arvense annotated genome (Nunn et al., \u0026nbsp;2022) was used for identification of the gene ID.\u003c/p\u003e","description":"","filename":"Fig.S1.docx","url":"https://assets-eu.researchsquare.com/files/rs-3221172/v1/f7905ad2fa7cbdee49d49371.docx"},{"id":41157877,"identity":"e3f93194-b284-43ff-a736-88a2926ef171","added_by":"auto","created_at":"2023-08-07 06:45:54","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":136070,"visible":true,"origin":"","legend":"\u003cp\u003eFig. S2. Expression profiling of individual genes and isoforms involved in oil bodies \u0026nbsp;formation during seed maturation by RNA-Seq (light grey bars) and qPCR (dark grey \u0026nbsp;bars). For RNA-Seq data, expression levels represented by FPKM values were \u0026nbsp;normalized to GREEN stage. The genes analysed, OLE1, OLE2, and OBA1a, are \u0026nbsp;indicated in each figure. For qPCR analysis, data were obtained from three independent \u0026nbsp;pools of seeds from five plants of each line. Data represent means of at least three \u0026nbsp;biological replicates.\u003c/p\u003e","description":"","filename":"Fig.S2.docx","url":"https://assets-eu.researchsquare.com/files/rs-3221172/v1/d3d6baaa0a474b279768d416.docx"},{"id":41158027,"identity":"9b9c0cfb-4036-4f45-8188-c68a0370af85","added_by":"auto","created_at":"2023-08-07 06:53:54","extension":"docx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":3034136,"visible":true,"origin":"","legend":"\u003cp\u003eFig. S3. HPTLC chromatograms of standards at UV 190 nm: pic1: 1-Oleoyl-rac-glycerol, \u0026nbsp;pic 2: 1,2-dioleoyl-rac-glycerol, pic 3: linoleic acid, pic 4: oleic acid, pic 5: erucic acid, \u0026nbsp;pic 6: glyceryl trioleate, pic 7: methyl oleate, pic 8: cholesteryl stearate, and pic 9: \u0026nbsp;cholesteryl oleate.\u003c/p\u003e","description":"","filename":"Fig.S3.docx","url":"https://assets-eu.researchsquare.com/files/rs-3221172/v1/3f1320c000e8ede123df2203.docx"},{"id":41157883,"identity":"4b5a2500-3993-421e-842c-5b56f6d07477","added_by":"auto","created_at":"2023-08-07 06:45:55","extension":"docx","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":14261371,"visible":true,"origin":"","legend":"\u003cp\u003eFig. S4. HPTLC chromatograms corresponding to samples at the different maturation \u0026nbsp;stages detected at UV 190 nm: a) GREEN, b) GREENYELLOW, c) YELLOWGREEN; \u0026nbsp;d) YELLOW, and e) MATURE.\u003c/p\u003e","description":"","filename":"Fig.S4.docx","url":"https://assets-eu.researchsquare.com/files/rs-3221172/v1/9230ea42a0e43658b635fcdc.docx"},{"id":41157882,"identity":"775c5d6e-a204-48f1-b26a-b7ec4293cede","added_by":"auto","created_at":"2023-08-07 06:45:55","extension":"docx","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":7782942,"visible":true,"origin":"","legend":"\u003cp\u003eFig. S5. HPTLC-ESI+ -MS/MS of molecular species found in separated and extracted \u0026nbsp;TAG peak (907.8, 935.9, 959.9, 989.9 and 1046.0)\u003c/p\u003e","description":"","filename":"Fig.S5.docx","url":"https://assets-eu.researchsquare.com/files/rs-3221172/v1/fa9b2f8f2dc01e6b9f941cd3.docx"},{"id":41157878,"identity":"94ba240f-4947-40fa-9574-115ce59f28a1","added_by":"auto","created_at":"2023-08-07 06:45:54","extension":"docx","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":14223,"visible":true,"origin":"","legend":"\u003cp\u003eTable S1 primers used for qPCR analysis in this study.\u003c/p\u003e","description":"","filename":"tableS1primers.docx","url":"https://assets-eu.researchsquare.com/files/rs-3221172/v1/d1a856695035fc6d77ba48fc.docx"},{"id":41157880,"identity":"d49fcfa9-b056-45e7-b325-da9e3569b2c1","added_by":"auto","created_at":"2023-08-07 06:45:54","extension":"xlsx","order_by":8,"title":"","display":"","copyAsset":false,"role":"supplement","size":10562,"visible":true,"origin":"","legend":"\u003cp\u003eTable S2. Total reads, clean reads and quality parameters of the RNA-Seq data.\u003c/p\u003e","description":"","filename":"TableS2.QCdatastatistics.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-3221172/v1/5fb02f03453f65ffdef079d2.xlsx"},{"id":41157884,"identity":"1c1f6d41-2a48-4c2e-8188-e4e508dba904","added_by":"auto","created_at":"2023-08-07 06:45:55","extension":"xlsx","order_by":9,"title":"","display":"","copyAsset":false,"role":"supplement","size":23886715,"visible":true,"origin":"","legend":"\u003cp\u003eSupporting information 1: RNA-Seq data set containing all the results for each \u0026nbsp;pairwise comparison used in this study.\u003c/p\u003e","description":"","filename":"SeedmaturationstagesRNAseqdeg.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-3221172/v1/5e6e4188b864bb4f8b6aaa7b.xlsx"}],"financialInterests":"","formattedTitle":"Integrative transcriptomic and HPTL-ESI-MS analysis reveals a strong coordination between genes and pathways contributing to high erucic triacylglycerol biosynthesis during Pennycress (Thlaspi arvense L.) seed maturation","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":"Thlaspi arvense, seed, oil, TAG, DGAT, PDAT, VLCFAs, erucic acid.","lastPublishedDoi":"10.21203/rs.3.rs-3221172/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3221172/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cem\u003eThlaspi arvense\u003c/em\u003e (Pennycress) is an emerging feedstock for biofuel production because of its high seed-oil content enriched in erucic acid (35-40% of total fatty acids). A transcriptomic analysis together with an HPTLC-ESI-MS and MS\u003csup\u003en\u003c/sup\u003e positional analysis characterization of the TAG fractions in five different seed maturation stages was performed to study the high erucic seed-oil biosynthesis in Pennycress. The RNA-Seq data showed a temporal pattern of regulation in which genes involved in the synthesis of carbon precursors and fatty acids were highly abundant at the earlier stages of maturation, then decreasing in the mature ones. Genes involved in TAG biosynthesis showed a complex temporal regulation in which \u003cem\u003eDGAT2\u003c/em\u003e and \u003cem\u003ePDAT1\u003c/em\u003e showed higher expression at the earlier stages of maturation, coincident with the rapid incorporation of 22:1 to TAG. Genes from the acyl-editing pathway showed higher expression at the earlier stages of seed maturation, suggesting a major role of this pathway at the initial stages of seed-oil biosynthesis. HPTLC-ESI-MS analysis showed an increase of TAG species containing VLCFAs (22:1 and 24:1) with 18:2 at \u003cem\u003esn-2\u003c/em\u003e position with seed maturation. This increase was concomitant with an increase of \u003cem\u003eDGAT1\u003c/em\u003e and \u003cem\u003ePDAT2\u003c/em\u003e mRNA levels, suggesting a higher Kennedy pathway activity. However, the concerted regulation between DGAT and PDAT enzymes suggested a coordination between pathways, isoforms and substrate pools for the synthesis of high erucic TAG in Pennycress.\u003c/p\u003e","manuscriptTitle":"Integrative transcriptomic and HPTL-ESI-MS analysis reveals a strong coordination between genes and pathways contributing to high erucic triacylglycerol biosynthesis during Pennycress (Thlaspi arvense L.) seed maturation","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-08-07 06:45:49","doi":"10.21203/rs.3.rs-3221172/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":"7e4e9304-5bc3-4500-96a1-273f58b6d1a2","owner":[],"postedDate":"August 7th, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2023-09-16T19:45:11+00:00","versionOfRecord":[],"versionCreatedAt":"2023-08-07 06:45:49","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3221172","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3221172","identity":"rs-3221172","version":["v1"]},"buildId":"_2-kVJe1T_tPrBINL-cwx","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}