PIF4-mediated thermomorphogenesis relies on its oligomerization ability, not DNA-binding or transactivation activity

PIF4-mediated thermomorphogenesis relies on its oligomerization ability, not DNA-binding or transactivation activity | 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 Article PIF4-mediated thermomorphogenesis relies on its oligomerization ability, not DNA-binding or transactivation activity Yongjian Qiu, Haibo Xiong, Abhishesh Bajracharya, Ranjeeta Odari, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7294532/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 17 Mar, 2026 Read the published version in Nature Communications → Version 1 posted You are reading this latest preprint version Abstract Plants tailor their architecture to warm ambient temperatures through the central thermosensory transcription factor PHYTOCHROME-INTERACTING FACTOR 4 (PIF4), yet the sequence features that confer this activity remain poorly defined. Here, we combine targeted mutagenesis, phase-separation assays, and transgenic complementation to dissect how structured and disordered regions of PIF4 contribute to its function in thermomorphogenesis. A long N-terminal intrinsically disordered region (IDR) enables PIF4 to form gel-like condensates in vitro and in planta . Within this IDR, we identify an acidic transactivation domain (TAD) and an extended basic segment that carries a nuclear-localization signal and the canonical basic motif of the basic helix-loop-helix (bHLH) domain. The basic segment is both necessary and sufficient to drive PIF4 condensate formation, while the TAD merely tunes condensate properties. Strikingly, alanine substitutions that abolish TAD-mediated transactivation, disrupt DNA binding, or greatly reduce phase-separation propensity have no significant effect on thermomorphogenetic hypocotyl elongation. By contrast, replacing three conserved basic residues in the first helix of the HLH domain disrupts PIF4 oligomerization and abolishes thermo-induced hypocotyl growth. We conclude that the ability of PIF4 to oligomerize, rather than to bind DNA or recruit the transcriptional machinery, is the primary determinant of its thermomorphogenetic activity. Biological sciences/Plant sciences/Plant molecular biology Biological sciences/Plant sciences/Plant signalling Biological sciences/Molecular biology/Transcription Thermomorphogenesis Phytochrome-Interacting Factor 4 (PIF4) Basic helix-loop-helix (bHLH) Transcriptional activation domain (TAD) DNA-binding domain (DBD) Intrinsically disordered region (IDR) Phase separation Full Text Additional Declarations There is NO Competing Interest. Supplementary Files TableS1.pdf Supplementary Table 1 TableS2.pdf Supplementary Table 2 SupplementaryFigures.pdf Supplementary Figures Cite Share Download PDF Status: Published Journal Publication published 17 Mar, 2026 Read the published version in Nature Communications → 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-7294532","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":508088526,"identity":"37853bbd-32d2-4e23-98a5-985d9de23ffa","order_by":0,"name":"Yongjian 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domain (TAD), DNA-binding domain (DBD), Intrinsically disordered region (IDR), Phase separation","lastPublishedDoi":"10.21203/rs.3.rs-7294532/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7294532/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Plants tailor their architecture to warm ambient temperatures through the central thermosensory transcription factor PHYTOCHROME-INTERACTING FACTOR 4 (PIF4), yet the sequence features that confer this activity remain poorly defined. Here, we combine targeted mutagenesis, phase-separation assays, and transgenic complementation to dissect how structured and disordered regions of PIF4 contribute to its function in thermomorphogenesis. A long N-terminal intrinsically disordered region (IDR) enables PIF4 to form gel-like condensates in vitro and \u003ci\u003ein planta\u003c/i\u003e. Within this IDR, we identify an acidic transactivation domain (TAD) and an extended basic segment that carries a nuclear-localization signal and the canonical basic motif of the basic helix-loop-helix (bHLH) domain. The basic segment is both necessary and sufficient to drive PIF4 condensate formation, while the TAD merely tunes condensate properties. Strikingly, alanine substitutions that abolish TAD-mediated transactivation, disrupt DNA binding, or greatly reduce phase-separation propensity have no significant effect on thermomorphogenetic hypocotyl elongation. By contrast, replacing three conserved basic residues in the first helix of the HLH domain disrupts PIF4 oligomerization and abolishes thermo-induced hypocotyl growth. 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