{"id":{"repo_id":"lancaster","oai_identifier":"oai:eprints.lancs.ac.uk:124536"},"canonical_url":"https://search.dev.ndltd.org/etd/lancaster/oai:eprints.lancs.ac.uk:124536","repository":{"repo_id":"lancaster","name":"Lancaster University","base_url":"https://eprints.lancs.ac.uk/cgi/oai2"},"display":{"title":"Using a chemical genetics approach to dissect the nitrogen signalling pathway in Arabidopsis","abstract":"Nitrate is an important nutrient and signalling molecule to plants. As it is taken up and assimilated, reduced forms of N accumulate and the expression of many genes associated with nitrate assimilation are repressed. Little is known about the mechanisms involved in this N repression. This project, for the first time, adopts a chemical genetics approach to investigate the feedback regulatory pathway that links the plant’s N status to expression of the NRT2.1 nitrate transporter gene. A novel chemical screening platform was developed that was designed to be used in conjunction with Arabidopsis lines expressing luciferase reporter genes in roots. This semi-hydroponic platform allows roots to be exposed to a variety of nutrient treatments in a 96-well plate format suitable for chemical genetic screens. This was combined with a newly developed ‘ice capture’ method that provided a rapid and efficient way to harvest root material for the luciferase assay. Using this screening platform in conjunction with a nitrate-inducible luciferase reporter line, pNRT2.1::LUC, three chemical libraries, containing 7420 bioactive molecules were screened in duplicate for compounds that antagonise N repression of luminescence. The screen identified a plant-derived alkaloid, camptothecin, that enhanced pNRT2.1::LUC expression under N-repressive conditions. The positive effect of camptothecin on expression of the endogenous NRT2.1 gene was confirmed using real-time PCR and shown to extend to other N-repressed genes of the nitrate assimilatory pathway. Camptothecin is known to target topoisomerase I, an enzyme that is increasingly being linked to a role in chromatin re-modelling, in addition to its more familiar roles in DNA replication and repair. The possible epigenetic role of topoisomerase I in repression of NRT2.1 and other genes of the nitrate assimilatory pathway is discussed. It was also observed that an arginine treatment strongly stimulated pNRT2.1::LUC in the luciferase assay, in a nitrate-dependent manner. Since this effect was not observed at the mRNA level, it is hypothesised that arginine was acting on pNRT2.1 expression at a post-transcriptional level.","abstract_html":"Nitrate is an important nutrient and signalling molecule to plants. As it is taken up and assimilated, reduced forms of N accumulate and the expression of many genes associated with nitrate assimilation are repressed. Little is known about the mechanisms involved in this N repression. This project, for the first time, adopts a chemical genetics approach to investigate the feedback regulatory pathway that links the plant’s N status to expression of the NRT2.1 nitrate transporter gene. A novel chemical screening platform was developed that was designed to be used in conjunction with Arabidopsis lines expressing luciferase reporter genes in roots. This semi-hydroponic platform allows roots to be exposed to a variety of nutrient treatments in a 96-well plate format suitable for chemical genetic screens. This was combined with a newly developed ‘ice capture’ method that provided a rapid and efficient way to harvest root material for the luciferase assay. Using this screening platform in conjunction with a nitrate-inducible luciferase reporter line, pNRT2.1::LUC, three chemical libraries, containing 7420 bioactive molecules were screened in duplicate for compounds that antagonise N repression of luminescence. The screen identified a plant-derived alkaloid, camptothecin, that enhanced pNRT2.1::LUC expression under N-repressive conditions. The positive effect of camptothecin on expression of the endogenous NRT2.1 gene was confirmed using real-time PCR and shown to extend to other N-repressed genes of the nitrate assimilatory pathway. Camptothecin is known to target topoisomerase I, an enzyme that is increasingly being linked to a role in chromatin re-modelling, in addition to its more familiar roles in DNA replication and repair. The possible epigenetic role of topoisomerase I in repression of NRT2.1 and other genes of the nitrate assimilatory pathway is discussed. It was also observed that an arginine treatment strongly stimulated pNRT2.1::LUC in the luciferase assay, in a nitrate-dependent manner. Since this effect was not observed at the mRNA level, it is hypothesised that arginine was acting on pNRT2.1 expression at a post-transcriptional level.","abstract_has_math":false,"creators":["Gent, Lucas","Forde, Brian","Hawkesford, Malcolm"],"institution":"Lancaster University","degree_name":"Ph.D.","degree_level":"doctoral","degree_discipline":null,"degree_department":null,"school":null,"contributors":[],"advisors":[],"committee_chairs":[],"committee_members":[],"year":2018,"date_issued":"2018","date_published":"2018","updated_at":"2026-07-24T02:48:43Z","subjects":[],"languages":[],"rights":[],"rights_urls":[],"identifier_entries":[]},"links":{"outbound_url":null,"outbound_label":null,"outbound_source":null},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:creator","label":"Author","values":["Gent, Lucas","Forde, Brian","Hawkesford, Malcolm"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date","label":"Dc Date","values":["2018"]},{"key":"dc:date.issued","label":"Date","values":["2018"]},{"key":"dc:publisher.commercial","label":"Dc Publisher Commercial","values":["Lancaster University"]},{"key":"dc:publisher.department","label":"Dc Publisher Department","values":["Lancaster Environment Centre"]},{"key":"dc:publisher.institution","label":"Dc Publisher Institution","values":["Lancaster University"]},{"key":"dc:relation.isreferencedby","label":"Dc Relation Isreferencedby","values":["https://eprints.lancs.ac.uk/id/eprint/124536/"]},{"key":"dc:type","label":"Dc Type","values":["Thesis"]},{"key":"dc:type.qualificationlevel","label":"Dc Type Qualificationlevel","values":["doctoral"]},{"key":"dc:type.qualificationname","label":"Dc Type Qualificationname","values":["Ph.D."]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier.uri","label":"Identifier URI","values":["https://eprints.lancs.ac.uk/id/eprint/124536/1/2018gentphd.pdf"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description.abstract","label":"Abstract","values":["Nitrate is an important nutrient and signalling molecule to plants. As it is taken up and assimilated, reduced forms of N accumulate and the expression of many genes associated with nitrate assimilation are repressed. Little is known about the mechanisms involved in this N repression. This project, for the first time, adopts a chemical genetics approach to investigate the feedback regulatory pathway that links the plant’s N status to expression of the NRT2.1 nitrate transporter gene. A novel chemical screening platform was developed that was designed to be used in conjunction with Arabidopsis lines expressing luciferase reporter genes in roots. This semi-hydroponic platform allows roots to be exposed to a variety of nutrient treatments in a 96-well plate format suitable for chemical genetic screens. This was combined with a newly developed ‘ice capture’ method that provided a rapid and efficient way to harvest root material for the luciferase assay. Using this screening platform in conjunction with a nitrate-inducible luciferase reporter line, pNRT2.1::LUC, three chemical libraries, containing 7420 bioactive molecules were screened in duplicate for compounds that antagonise N repression of luminescence. The screen identified a plant-derived alkaloid, camptothecin, that enhanced pNRT2.1::LUC expression under N-repressive conditions. The positive effect of camptothecin on expression of the endogenous NRT2.1 gene was confirmed using real-time PCR and shown to extend to other N-repressed genes of the nitrate assimilatory pathway. Camptothecin is known to target topoisomerase I, an enzyme that is increasingly being linked to a role in chromatin re-modelling, in addition to its more familiar roles in DNA replication and repair. The possible epigenetic role of topoisomerase I in repression of NRT2.1 and other genes of the nitrate assimilatory pathway is discussed. It was also observed that an arginine treatment strongly stimulated pNRT2.1::LUC in the luciferase assay, in a nitrate-dependent manner. Since this effect was not observed at the mRNA level, it is hypothesised that arginine was acting on pNRT2.1 expression at a post-transcriptional level."]},{"key":"dc:format","label":"Dc Format","values":["application/pdf"]},{"key":"dc:title","label":"Title","values":["Using a chemical genetics approach to dissect the nitrogen signalling pathway in Arabidopsis"]}]}],"canonical_facts":{"dc:creator":["Gent, Lucas","Forde, Brian","Hawkesford, Malcolm"],"dc:date":["2018"],"dc:date.issued":["2018"],"dc:description.abstract":["Nitrate is an important nutrient and signalling molecule to plants. As it is taken up and assimilated, reduced forms of N accumulate and the expression of many genes associated with nitrate assimilation are repressed. Little is known about the mechanisms involved in this N repression. This project, for the first time, adopts a chemical genetics approach to investigate the feedback regulatory pathway that links the plant’s N status to expression of the NRT2.1 nitrate transporter gene. A novel chemical screening platform was developed that was designed to be used in conjunction with Arabidopsis lines expressing luciferase reporter genes in roots. This semi-hydroponic platform allows roots to be exposed to a variety of nutrient treatments in a 96-well plate format suitable for chemical genetic screens. This was combined with a newly developed ‘ice capture’ method that provided a rapid and efficient way to harvest root material for the luciferase assay. Using this screening platform in conjunction with a nitrate-inducible luciferase reporter line, pNRT2.1::LUC, three chemical libraries, containing 7420 bioactive molecules were screened in duplicate for compounds that antagonise N repression of luminescence. 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