The terminal phase of leaf senescence is gated by the cytosolic arginine pool

The terminal phase of leaf senescence is gated by the cytosolic arginine pool | 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 The terminal phase of leaf senescence is gated by the cytosolic arginine pool Olivier Keech, Shah Hussain, Clément Boussardon This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6205660/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted You are reading this latest preprint version Abstract Leaf senescence aims at degrading cell components to recover valuable nutrients, and reallocate them towards other organs2 (Fig 1a). Once this remobilisation is complete, cells undergo a vacuolar-type of programmed cell death2, ultimately leading to the death of the entire organ. But how do cells from a senescing leaf “know” when to die? If the cell death process per se is initiated too early, the remobilisation might not be complete and, hence futile. This suggests the presence of a “sensing” mechanism that coordinates the remobilization phase with the onset of cell death during leaf senescence (Fig 1a). Using functional stay-green mutants, we show that senescing cells are wired to metabolically dissipate the cytosolic arginine pool, which otherwise represses the progression of leaf senescence. We propose a model in which a senescing cell uses this pool as a proxy for the completion of the remobilisation of nitrogen, and to time accurately the subsequent induction of cell death. Biological sciences/Plant sciences/Plant stress responses/Abiotic Biological sciences/Plant sciences/Plant development/Cell fate Figures Figure 1 Figure 2 Full Text Additional Declarations There is NO Competing Interest. Supplementary Files SupplementaryFigure1.pdf Supp Figure 1 SupplementaryFigure2.pdf Supp Figure 2 SupplementaryFigure3.pdf Supp Figure 3 Cite Share Download PDF Status: Under Review 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-6205660","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Letter","associatedPublications":[],"authors":[{"id":431236544,"identity":"1afdd467-a534-4cee-8e86-8039f90ae210","order_by":0,"name":"Olivier Keech","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA3klEQVRIiWNgGAWjYDACdsYGhgQgLQHEzAwVxGhhRtFyhigtUBqshbGNCB38zMxtEg8q7jFItjcf/Fw473C0wQH2hw/waZFsZmyTSDhTzCDNcyxZeua2w7kbDvAYG+DTYnCYsdkgsS2BQU4ix4yZF6KFTYKwln9ALfLvvzHzzgFpYX/+g4CWxgeJDQkM0hI8bMy8DSAtDGb4dID80vgg4VgCj2RPmjHQP+m5Mw/zGON1GD97+4ODP2oS5CSOH374mafGOrfvePvDD3itgQIeBJMZt6pRMApGwSgYBUQCAMShRVkwaNfeAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0002-0546-7721","institution":"Umea University","correspondingAuthor":true,"prefix":"","firstName":"Olivier","middleName":"","lastName":"Keech","suffix":""},{"id":431236545,"identity":"be4e7acb-7c9d-4439-8d8d-a3ab642e9be3","order_by":1,"name":"Shah Hussain","email":"","orcid":"","institution":"Umea University","correspondingAuthor":false,"prefix":"","firstName":"Shah","middleName":"","lastName":"Hussain","suffix":""},{"id":431236546,"identity":"69da61cc-db4a-498a-aa14-5952b6aa0918","order_by":2,"name":"Clément Boussardon","email":"","orcid":"","institution":"Umea University","correspondingAuthor":false,"prefix":"","firstName":"Clément","middleName":"","lastName":"Boussardon","suffix":""}],"badges":[],"createdAt":"2025-03-11 17:50:37","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6205660/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6205660/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":78933874,"identity":"b541ed61-4389-4acb-9b10-c01d4cd2cc4e","added_by":"auto","created_at":"2025-03-21 04:04:30","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":253775,"visible":true,"origin":"","legend":"\u003cp\u003eThe cytosolic arginine pool is modulated by transporters induced in a \u0026nbsp;senescence-dependent manner. a, Schematic view of the leaf senescence program, \u0026nbsp;including the question addressed in this study. b, Chlorophyll content in darkened leaf discs \u0026nbsp;treated supplemented with amino acid (AA). Leaf discs were incubated in a buffered solution \u0026nbsp;containing 1mM of the indicated AA, and were kept for 6 days in darkness. Chlorophyll content \u0026nbsp;was quantified, and the results are mean ± S.D., n = 4 independent biological replicates. \u0026nbsp;Significance: two-tailed Student’s t-test with **p\u0026lt;0.01 and ***p\u0026lt;0.001. c, Schematic model \u0026nbsp;depicting the transport of arginine (arg) to the apoplast via AAP5 or to mitochondria via BAC1 \u0026nbsp;or BAC2, along with its associated metabolic pathways. d, Transcriptional levels of BAC1, \u0026nbsp;BAC2 and AAP5 in a control leaf (Light) and in a leaf individually darkened (IDL) for 6 or 9 \u0026nbsp;days. Data were obtained by qRT-PCR (primers found in Supplementary Table 1), and the \u0026nbsp;results are mean ± SD of 4 independent replicates. Significant differences between genotypes \u0026nbsp;were assessed using Student’s t-test with *p\u0026lt;0.05; **p\u0026lt;0.01; and ***p\u0026lt;0.001. e,f, Chromatin \u0026nbsp;immunoprecipitation (ChIP) assays. ORE1 directly binds the promoter of BAC2 but not of \u0026nbsp;AAP5. Schematic diagrams show 1.5 kb upstream sequence of the BAC2 and AAP5 promoters, with a translational start sites (ATG) indicated at position +1, respectively. The putative and preferred ORE1 binding sites in the P1 and P2 regions of BAC2 and AAP5 promoters were shown in black and green (e,f, top), respectively. ChIP analyses were \u0026nbsp;conducted using anti-GFP antibodies with chromatin extracted from young (Growth \u0026nbsp;stage#1.12˷1.14) and old (Growth stage#8.0034) ore1-2 and ore1-2/pORE1::ORE1-GFP#5 plants (see Supplementary Fig. 1c-d for an independent line). Subsequently, qPCR analysis \u0026nbsp;was performed using ChIP-DNA as template and primers (Supplementary Table 1) specifically \u0026nbsp;targeting the promoter regions of BAC2 (e; bottom) and AAP5 (f; bottom) genes. TUBULIN2 (TUB2) was used as a control. Data are represented as mean ± S.D., n = 4 independent \u0026nbsp;biological replicates. Different letters above the bars indicate statistically significant differences \u0026nbsp;(p\u0026lt; 0.05\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-6205660/v1/6988c331b6f4f54b1af3c4ff.png"},{"id":78933132,"identity":"79bbaf26-d93a-4007-b03a-72071102948e","added_by":"auto","created_at":"2025-03-21 03:48:30","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":583289,"visible":true,"origin":"","legend":"\u003cp\u003eDissipating the cytosolic arginine pool reactivates the progression of leaf \u0026nbsp;senescence. a, Phenotype of individually-darkened leaves of WT, pif5-621, ore1-2 and \u0026nbsp;complemented lines expressing UBI10::BAC1, UBI10::BAC2 or UBI10::AAP5 in WT, pif5-621 and ore1-2 background. Leaves were individually darkened for 6 (white arrowhead) or 9 (red \u0026nbsp;arrowhead) days. b, Chlorophyll in IDL of plants used in (a). Data are represented as mean ± \u0026nbsp;S.D., n = 4 independent biological replicates. Significant differences were determined by \u0026nbsp;Student's t-test (***p\u0026lt; 0.001) after comparison with WT (black asterisks), with pif5-621 (dark \u0026nbsp;green asterisks) or with ore1-2 (light green asterisks). c, Arginine abundance (pmol/mg fresh \u0026nbsp;weight (FW)) in WT, pif5-621, ore1-2, WT/UBI10::BAC2, pif5-621/UBI10::BAC2 and pif5- 621/UBI10::AAP5 leaves in light and individually darkened for 6 or 9 days. Data are presented \u0026nbsp;as mean values. ± S.D, n = 4 independent biological replicates. ***p\u0026lt; 0.001) after Student’s t-test compared with WT (black asterisks) or with pif5-621 (dark green asterisks). d, Working \u0026nbsp;model depicting the key role of the cytosolic arginine pool in gating the transition between the \u0026nbsp;remobilization and termination phases. The dynamics of this pool is genetically controlled by \u0026nbsp;ORE1, which directly promotes BAC2 transcription (solid arrow) and indirectly positively \u0026nbsp;regulates the expression of AAP5 gene (dashed arrow). Abbreviations: ORE1, Oresara1; \u0026nbsp;BAC2, Basic Amino Acid Carrier2; AAP5, Amino Acid Permease5\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-6205660/v1/e214fb6f9aef1bc6746e07e0.png"},{"id":78934577,"identity":"557ece96-5a0f-47c6-8a9a-b6cded82c172","added_by":"auto","created_at":"2025-03-21 04:12:32","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":655919,"visible":true,"origin":"","legend":"Article File","description":"","filename":"Hussainetal20250311.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6205660/v1_covered_2bd20142-5780-41cc-9213-ef4f94b4f25e.pdf"},{"id":78933686,"identity":"d024abf5-ccbf-407f-9615-aa62660e4d4b","added_by":"auto","created_at":"2025-03-21 03:56:30","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":201391,"visible":true,"origin":"","legend":"\u003cp\u003eSupp Figure 1\u003c/p\u003e","description":"","filename":"SupplementaryFigure1.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6205660/v1/477650e921a03626ce9b8e40.pdf"},{"id":78933141,"identity":"13816c2f-9ee4-4176-b453-a379a1283565","added_by":"auto","created_at":"2025-03-21 03:48:30","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":108068,"visible":true,"origin":"","legend":"\u003cp\u003eSupp Figure 2\u003c/p\u003e","description":"","filename":"SupplementaryFigure2.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6205660/v1/cf437cd2f15866aa35781e5e.pdf"},{"id":78933134,"identity":"b47e2f0e-cbf2-4ad0-b307-53a2219f098b","added_by":"auto","created_at":"2025-03-21 03:48:30","extension":"pdf","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":47738,"visible":true,"origin":"","legend":"\u003cp\u003eSupp Figure 3\u003c/p\u003e","description":"","filename":"SupplementaryFigure3.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6205660/v1/7499e9fb7c778965c7794eae.pdf"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"The terminal phase of leaf senescence is gated by the cytosolic arginine pool","fulltext":[],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":false,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"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":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-6205660/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6205660/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Leaf senescence aims at degrading cell components to recover valuable nutrients, and reallocate them towards other organs2 (Fig 1a). Once this remobilisation is complete, cells undergo a vacuolar-type of programmed cell death2, ultimately leading to the death of the entire organ. But how do cells from a senescing leaf “know” when to die? If the cell death process per se is initiated too early, the remobilisation might not be complete and, hence futile. This suggests the presence of a “sensing” mechanism that coordinates the remobilization phase with the onset of cell death during leaf senescence (Fig 1a). Using functional stay-green mutants, we show that senescing cells are wired to metabolically dissipate the cytosolic arginine pool, which otherwise represses the progression of leaf senescence. We propose a model in which a senescing cell uses this pool as a proxy for the completion of the remobilisation of nitrogen, and to time accurately the subsequent induction of cell death.","manuscriptTitle":"The terminal phase of leaf senescence is gated by the cytosolic arginine pool","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-03-21 03:48:25","doi":"10.21203/rs.3.rs-6205660/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-plants","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"nplants","sideBox":"Learn more about [Nature Plants](http://www.nature.com/nplants/)","snPcode":"","submissionUrl":"","title":"Nature Plants","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Research","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"29736a20-d650-421f-b7b2-bdf1820d5d14","owner":[],"postedDate":"March 21st, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":45933286,"name":"Biological sciences/Plant sciences/Plant stress responses/Abiotic"},{"id":45933287,"name":"Biological sciences/Plant sciences/Plant development/Cell fate"}],"tags":[],"updatedAt":"2026-05-12T07:43:51+00:00","versionOfRecord":[],"versionCreatedAt":"2025-03-21 03:48:25","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6205660","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6205660","identity":"rs-6205660","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}