{"id":{"repo_id":"auckland-ms","oai_identifier":"oai:researchspace.auckland.ac.nz:2292/71109"},"canonical_url":"https://search.dev.ndltd.org/etd/auckland-ms/oai:researchspace.auckland.ac.nz:2292/71109","repository":{"repo_id":"auckland-ms","name":"University of Auckland","base_url":"https://researchspace.auckland.ac.nz/server/oai/request"},"display":{"title":"Investigating key regulators of nitrogen demand signalling and symbiotic nitrogen fixation in Medicago truncatula","abstract":"Nitrogen (N) is an essential nutrient required for plant growth and represents a primary limiting factor in determining crop yield. To overcome N limitation, legumes form mutualistic relationship with N-fixing bacteria resulting in the formation of root nodules, where symbiotic N-fixation (SNF) occurs. The formation of nodules is an energy expensive process. Hence, nodulation in legumes is strictly regulated by the host through a peptide-receptor mediated systemic negative feedback loop known as Autoregulation of Nodulation (AON). However, how AON controls nodulation and what are the molecular components of AON is not well understood. In this thesis, I have identified and characterised downstream targets of CLE-SUNN mediated AON pathway in the model legume Medicago truncatula. RNA sequencing of CLE13-TaP treated M. truncatula plants revealed numerous genes potentially involved in nodulation. Agrobacterium rhizogenes-mediated reverse genetic approaches were carried out for significantly upregulated genes (MYB102, WRKY70A and SUPERROOT2) and downregulated genes (bHLH84 and ZFP6) in order to elucidate their role in nodulation. I have shown that overexpression of upregulated genes decreased the nodule number, while overexpression of downregulated target genes increased the nodule number. Moreover, overexpression of MYB102, WRKY70A, SUPERROOT2 and knockdown of bHLH84 & ZFP6 resulted in significant decrease in nodule number in the sunn4 mutant that is defective in AON. Therefore, these selected target genes likely are downstream of SUNN receptor and play crucial roles in CLE-SUNN mediated AON pathway. However, further analysis is necessary to strengthen our understanding of the specific role of these genes in AON signalling. In order to examine the influence of ethylene signalling, a local regulator of nodulation on the systemic CLE-SUNN mediated AON signalling, both the wild type and ethylene-insensitive skl mutant were subjected to treatment with ACC, which is a precursor of the ethylene biosynthetic pathway. qRT-PCR analysis of 24 hours CLE13-TaP treated and ACC treated plants showed a significant downregulation of several LysM-RLKs and NF-induced genes. These suggest a possible convergence between local ethylene signalling and systemic CLE-SUNN mediated AON signalling to regulate legume-rhizobial symbiosis. Conclusively, the results presented in this thesis provide insights into how nodulation is regulated in M. truncatula.","abstract_html":"Nitrogen (N) is an essential nutrient required for plant growth and represents a primary limiting factor in determining crop yield. To overcome N limitation, legumes form mutualistic relationship with N-fixing bacteria resulting in the formation of root nodules, where symbiotic N-fixation (SNF) occurs. The formation of nodules is an energy expensive process. Hence, nodulation in legumes is strictly regulated by the host through a peptide-receptor mediated systemic negative feedback loop known as Autoregulation of Nodulation (AON). However, how AON controls nodulation and what are the molecular components of AON is not well understood. In this thesis, I have identified and characterised downstream targets of CLE-SUNN mediated AON pathway in the model legume Medicago truncatula. RNA sequencing of CLE13-TaP treated M. truncatula plants revealed numerous genes potentially involved in nodulation. Agrobacterium rhizogenes-mediated reverse genetic approaches were carried out for significantly upregulated genes (MYB102, WRKY70A and SUPERROOT2) and downregulated genes (bHLH84 and ZFP6) in order to elucidate their role in nodulation. I have shown that overexpression of upregulated genes decreased the nodule number, while overexpression of downregulated target genes increased the nodule number. Moreover, overexpression of MYB102, WRKY70A, SUPERROOT2 and knockdown of bHLH84 & ZFP6 resulted in significant decrease in nodule number in the sunn4 mutant that is defective in AON. Therefore, these selected target genes likely are downstream of SUNN receptor and play crucial roles in CLE-SUNN mediated AON pathway. However, further analysis is necessary to strengthen our understanding of the specific role of these genes in AON signalling. In order to examine the influence of ethylene signalling, a local regulator of nodulation on the systemic CLE-SUNN mediated AON signalling, both the wild type and ethylene-insensitive skl mutant were subjected to treatment with ACC, which is a precursor of the ethylene biosynthetic pathway. qRT-PCR analysis of 24 hours CLE13-TaP treated and ACC treated plants showed a significant downregulation of several LysM-RLKs and NF-induced genes. These suggest a possible convergence between local ethylene signalling and systemic CLE-SUNN mediated AON signalling to regulate legume-rhizobial symbiosis. Conclusively, the results presented in this thesis provide insights into how nodulation is regulated in M. truncatula.","abstract_has_math":false,"creators":["Banda, Sushmitha"],"institution":"ResearchSpace@Auckland","degree_name":"PhD","degree_level":"Doctoral","degree_discipline":"Biological Sciences","degree_department":null,"school":null,"contributors":[],"advisors":["Macinnis-Ng, Cate","Imin, Nijat","David, Karine"],"committee_chairs":[],"committee_members":[],"year":2024,"date_issued":"2024","date_published":"2024","updated_at":"2026-07-24T01:02:40Z","subjects":["Hairy root transformation","Reverse genetics","N-fixation","Autoregulation of nodulation","Plant molecular science"],"languages":[],"rights":["Items in ResearchSpace are protected by copyright, with all rights reserved, unless otherwise indicated."],"rights_urls":["https://researchspace.auckland.ac.nz/docs/uoa-docs/rights.htm"],"identifier_entries":[]},"links":{"outbound_url":"https://hdl.handle.net/2292/71109","outbound_label":"Handle","outbound_source":"dc:identifier.uri"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor.advisor","label":"Advisor","values":["Macinnis-Ng, Cate","Imin, Nijat","David, Karine"]},{"key":"dc:creator","label":"Author","values":["Banda, Sushmitha"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date.accessioned","label":"Dc Date Accessioned","values":["2025-02-03T19:06:25Z"]},{"key":"dc:date.available","label":"Dc Date Available","values":["2025-02-03T19:06:25Z"]},{"key":"dc:date.issued","label":"Date","values":["2024"]},{"key":"dc:publisher","label":"Institution","values":["ResearchSpace@Auckland"]},{"key":"dc:type","label":"Dc Type","values":["Thesis"]},{"key":"thesis:degree_discipline","label":"Discipline","values":["Biological Sciences"]},{"key":"thesis:degree_level","label":"Degree Level","values":["Doctoral"]},{"key":"thesis:degree_name","label":"Degree Name","values":["PhD"]},{"key":"thesis:institution_name","label":"Thesis Institution Name","values":["The University of Auckland"]}]},{"id":"subjects_keywords","label":"Subjects and Keywords","entries":[{"key":"dc:subject","label":"Dc Subject","values":["Hairy root transformation","Reverse genetics","N-fixation","Autoregulation of nodulation","Plant molecular science"]}]},{"id":"language_rights","label":"Language and Rights","entries":[{"key":"dc:rights","label":"Dc Rights","values":["Items in ResearchSpace are protected by copyright, with all rights reserved, unless otherwise indicated."]},{"key":"dc:rights.uri","label":"Rights URI","values":["https://researchspace.auckland.ac.nz/docs/uoa-docs/rights.htm"]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier.uri","label":"Identifier URI","values":["https://hdl.handle.net/2292/71109"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description.abstract","label":"Abstract","values":["Nitrogen (N) is an essential nutrient required for plant growth and represents a primary limiting factor in determining crop yield. To overcome N limitation, legumes form mutualistic relationship with N-fixing bacteria resulting in the formation of root nodules, where symbiotic N-fixation (SNF) occurs. The formation of nodules is an energy expensive process. Hence, nodulation in legumes is strictly regulated by the host through a peptide-receptor mediated systemic negative feedback loop known as Autoregulation of Nodulation (AON). However, how AON controls nodulation and what are the molecular components of AON is not well understood. In this thesis, I have identified and characterised downstream targets of CLE-SUNN mediated AON pathway in the model legume Medicago truncatula. RNA sequencing of CLE13-TaP treated M. truncatula plants revealed numerous genes potentially involved in nodulation. Agrobacterium rhizogenes-mediated reverse genetic approaches were carried out for significantly upregulated genes (MYB102, WRKY70A and SUPERROOT2) and downregulated genes (bHLH84 and ZFP6) in order to elucidate their role in nodulation. I have shown that overexpression of upregulated genes decreased the nodule number, while overexpression of downregulated target genes increased the nodule number. Moreover, overexpression of MYB102, WRKY70A, SUPERROOT2 and knockdown of bHLH84 & ZFP6 resulted in significant decrease in nodule number in the sunn4 mutant that is defective in AON. Therefore, these selected target genes likely are downstream of SUNN receptor and play crucial roles in CLE-SUNN mediated AON pathway. However, further analysis is necessary to strengthen our understanding of the specific role of these genes in AON signalling. In order to examine the influence of ethylene signalling, a local regulator of nodulation on the systemic CLE-SUNN mediated AON signalling, both the wild type and ethylene-insensitive skl mutant were subjected to treatment with ACC, which is a precursor of the ethylene biosynthetic pathway. qRT-PCR analysis of 24 hours CLE13-TaP treated and ACC treated plants showed a significant downregulation of several LysM-RLKs and NF-induced genes. These suggest a possible convergence between local ethylene signalling and systemic CLE-SUNN mediated AON signalling to regulate legume-rhizobial symbiosis. Conclusively, the results presented in this thesis provide insights into how nodulation is regulated in M. truncatula."]},{"key":"dc:title","label":"Title","values":["Investigating key regulators of nitrogen demand signalling and symbiotic nitrogen fixation in Medicago truncatula"]}]}],"canonical_facts":{"dc:contributor.advisor":["Macinnis-Ng, Cate","Imin, Nijat","David, Karine"],"dc:creator":["Banda, Sushmitha"],"dc:date.accessioned":["2025-02-03T19:06:25Z"],"dc:date.available":["2025-02-03T19:06:25Z"],"dc:date.issued":["2024"],"dc:description.abstract":["Nitrogen (N) is an essential nutrient required for plant growth and represents a primary limiting factor in determining crop yield. To overcome N limitation, legumes form mutualistic relationship with N-fixing bacteria resulting in the formation of root nodules, where symbiotic N-fixation (SNF) occurs. The formation of nodules is an energy expensive process. Hence, nodulation in legumes is strictly regulated by the host through a peptide-receptor mediated systemic negative feedback loop known as Autoregulation of Nodulation (AON). However, how AON controls nodulation and what are the molecular components of AON is not well understood. In this thesis, I have identified and characterised downstream targets of CLE-SUNN mediated AON pathway in the model legume Medicago truncatula. RNA sequencing of CLE13-TaP treated M. truncatula plants revealed numerous genes potentially involved in nodulation. Agrobacterium rhizogenes-mediated reverse genetic approaches were carried out for significantly upregulated genes (MYB102, WRKY70A and SUPERROOT2) and downregulated genes (bHLH84 and ZFP6) in order to elucidate their role in nodulation. I have shown that overexpression of upregulated genes decreased the nodule number, while overexpression of downregulated target genes increased the nodule number. Moreover, overexpression of MYB102, WRKY70A, SUPERROOT2 and knockdown of bHLH84 & ZFP6 resulted in significant decrease in nodule number in the sunn4 mutant that is defective in AON. Therefore, these selected target genes likely are downstream of SUNN receptor and play crucial roles in CLE-SUNN mediated AON pathway. However, further analysis is necessary to strengthen our understanding of the specific role of these genes in AON signalling. In order to examine the influence of ethylene signalling, a local regulator of nodulation on the systemic CLE-SUNN mediated AON signalling, both the wild type and ethylene-insensitive skl mutant were subjected to treatment with ACC, which is a precursor of the ethylene biosynthetic pathway. qRT-PCR analysis of 24 hours CLE13-TaP treated and ACC treated plants showed a significant downregulation of several LysM-RLKs and NF-induced genes. These suggest a possible convergence between local ethylene signalling and systemic CLE-SUNN mediated AON signalling to regulate legume-rhizobial symbiosis. Conclusively, the results presented in this thesis provide insights into how nodulation is regulated in M. truncatula."],"dc:identifier.uri":["https://hdl.handle.net/2292/71109"],"dc:publisher":["ResearchSpace@Auckland"],"dc:rights":["Items in ResearchSpace are protected by copyright, with all rights reserved, unless otherwise indicated."],"dc:rights.uri":["https://researchspace.auckland.ac.nz/docs/uoa-docs/rights.htm"],"dc:subject":["Hairy root transformation","Reverse genetics","N-fixation","Autoregulation of nodulation","Plant molecular science"],"dc:title":["Investigating key regulators of nitrogen demand signalling and symbiotic nitrogen fixation in Medicago truncatula"],"dc:type":["Thesis"],"thesis:degree_discipline":["Biological Sciences"],"thesis:degree_level":["Doctoral"],"thesis:degree_name":["PhD"],"thesis:institution_name":["The University of Auckland"]},"updated_at":"2026-07-24T01:02:40Z"}