{"id":{"repo_id":"vt","oai_identifier":"oai:vtechworks.lib.vt.edu:10919/135468"},"canonical_url":"https://search.dev.ndltd.org/etd/vt/oai:vtechworks.lib.vt.edu:10919/135468","repository":{"repo_id":"vt","name":"Virginia Tech","base_url":"https://vtechworks.lib.vt.edu/oai/request"},"display":{"title":"Decoding the Transcriptional Specificity of Auxin Signaling: A Synthetic Biology Approach","abstract":"Auxin is a central regulator of nearly all aspects of plant growth, development, and environmental response. Despite its broad roles, the mechanisms by which a single hormone elicits such diverse and context-specific transcriptional responses remain unclear. These responses are primarily mediated by the nuclear auxin signaling pathway, which consists of ARF transcriptional activators, Aux/IAA repressors, and TIR1/AFB auxin receptors that work together to modulate gene expression. Functional diversity within these modular components, such as differences in DNA binding, protein-protein interactions, subcellular localization, and co-expression patterns is believed to act in concert to tune auxin signaling specificity. In support of our efforts to decode auxin signaling specificity, we developed an open-source, R-based analysis pipeline to process flow cytometry data generated by protoplast transient expression assays. This reproducible workflow automates transformation-based reporter analysis, streamlining quantification of gene expression and effector function in high-throughput experiments. To understand the functional diversity in ARF-mediated transcriptional responses, we performed RNA sequencing of Arabidopsis protoplasts transiently expressing irrepressible ARF variants to determine if they have distinct downstream regulatory targets. Transcriptomic analysis revealed that while each ARF appears to regulate largely overlapping sets of genes, they do so with varying efficiencies. Gene clusters preferentially regulated by specific ARFs were associated with distinct biological processes, aligning with the known developmental roles of those specific ARFs. While our transcriptomic analysis suggested that ARFs can drive distinct transcriptional outputs independently of Aux/IAAs, the extent to which specific ARF-Aux/IAA interactions contribute to this specificity remained unclear. To determine whether specific ARF-Aux/IAA interactions also contribute to transcriptional specificity, we developed a synthetic system that isolates these interactions using a recombinant protein-interaction domain from an animal ortholog. The system was validated through structural prediction, yeast two-hybrid assays, and protoplast transient expression assays. This system serves as the closest approximation to date of native ARF-Aux/IAA interactions, providing a powerful tool to dissect signaling specificity at the level of individual protein pairings. Together, the development of a streamlined data analysis pipeline, transcriptional profiling, and a synthetic interaction system provides both computational and experimental tools that advance our understanding of auxin signaling specificity. Ultimately, these insights will help explain how a single hormone can direct a vast array of developmental and environmental responses in plants.","abstract_html":"Auxin is a central regulator of nearly all aspects of plant growth, development, and environmental response. Despite its broad roles, the mechanisms by which a single hormone elicits such diverse and context-specific transcriptional responses remain unclear. These responses are primarily mediated by the nuclear auxin signaling pathway, which consists of ARF transcriptional activators, Aux/IAA repressors, and TIR1/AFB auxin receptors that work together to modulate gene expression. Functional diversity within these modular components, such as differences in DNA binding, protein-protein interactions, subcellular localization, and co-expression patterns is believed to act in concert to tune auxin signaling specificity. In support of our efforts to decode auxin signaling specificity, we developed an open-source, R-based analysis pipeline to process flow cytometry data generated by protoplast transient expression assays. This reproducible workflow automates transformation-based reporter analysis, streamlining quantification of gene expression and effector function in high-throughput experiments. To understand the functional diversity in ARF-mediated transcriptional responses, we performed RNA sequencing of Arabidopsis protoplasts transiently expressing irrepressible ARF variants to determine if they have distinct downstream regulatory targets. Transcriptomic analysis revealed that while each ARF appears to regulate largely overlapping sets of genes, they do so with varying efficiencies. Gene clusters preferentially regulated by specific ARFs were associated with distinct biological processes, aligning with the known developmental roles of those specific ARFs. While our transcriptomic analysis suggested that ARFs can drive distinct transcriptional outputs independently of Aux/IAAs, the extent to which specific ARF-Aux/IAA interactions contribute to this specificity remained unclear. To determine whether specific ARF-Aux/IAA interactions also contribute to transcriptional specificity, we developed a synthetic system that isolates these interactions using a recombinant protein-interaction domain from an animal ortholog. The system was validated through structural prediction, yeast two-hybrid assays, and protoplast transient expression assays. This system serves as the closest approximation to date of native ARF-Aux/IAA interactions, providing a powerful tool to dissect signaling specificity at the level of individual protein pairings. Together, the development of a streamlined data analysis pipeline, transcriptional profiling, and a synthetic interaction system provides both computational and experimental tools that advance our understanding of auxin signaling specificity. Ultimately, these insights will help explain how a single hormone can direct a vast array of developmental and environmental responses in plants.","abstract_has_math":false,"creators":["Taylor, Joseph Sylvester"],"institution":"Virginia Tech","degree_name":"Doctor of Philosophy","degree_level":"doctoral","degree_discipline":"Horticulture","degree_department":"Horticulture","school":null,"contributors":[],"advisors":[],"committee_chairs":["Bargmann, Bastiaan"],"committee_members":["Wang, Xiaofeng","Winkel, Brenda Sophia J.","Wright, Robert Clay"],"year":2025,"date_issued":"2025-06-10","date_published":"2025-06-10","updated_at":"2026-07-22T22:18:47Z","subjects":["auxin","plant development","synthetic biology","protoplast","transient expression"],"languages":["en"],"rights":["In Copyright"],"rights_urls":["http://rightsstatements.org/vocab/InC/1.0/"],"identifier_entries":[{"key":"dc:identifier.other","label":"Dc Identifier Other","values":["vt_gsexam:44070"],"render_values":[{"text":"vt_gsexam:44070","href":null,"code":true}]}]},"links":{"outbound_url":"https://hdl.handle.net/10919/135468","outbound_label":"Handle","outbound_source":"dc:identifier.uri"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor.committeechair","label":"Committee Chair","values":["Bargmann, Bastiaan"]},{"key":"dc:contributor.committeemember","label":"Committee Member","values":["Wang, Xiaofeng","Winkel, Brenda Sophia J.","Wright, Robert Clay"]},{"key":"dc:contributor.department","label":"Department","values":["Horticulture"]},{"key":"dc:creator","label":"Author","values":["Taylor, Joseph Sylvester"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date.accessioned","label":"Dc Date Accessioned","values":["2025-06-11T08:02:57Z"]},{"key":"dc:date.available","label":"Dc Date Available","values":["2025-06-11T08:02:57Z"]},{"key":"dc:date.issued","label":"Date","values":["2025-06-10"]},{"key":"dc:publisher","label":"Institution","values":["Virginia Tech"]},{"key":"dc:type","label":"Dc Type","values":["Dissertation"]},{"key":"thesis:degree_discipline","label":"Discipline","values":["Horticulture"]},{"key":"thesis:degree_level","label":"Degree Level","values":["doctoral"]},{"key":"thesis:degree_name","label":"Degree Name","values":["Doctor of Philosophy"]},{"key":"thesis:institution_name","label":"Thesis Institution Name","values":["Virginia Polytechnic Institute and State University"]}]},{"id":"subjects_keywords","label":"Subjects and Keywords","entries":[{"key":"dc:subject","label":"Dc Subject","values":["auxin","plant development","synthetic biology","protoplast","transient expression"]}]},{"id":"language_rights","label":"Language and Rights","entries":[{"key":"dc:language.iso","label":"Language (ISO)","values":["en"]},{"key":"dc:rights","label":"Dc Rights","values":["In Copyright"]},{"key":"dc:rights.uri","label":"Rights URI","values":["http://rightsstatements.org/vocab/InC/1.0/"]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier.other","label":"Dc Identifier Other","values":["vt_gsexam:44070"]},{"key":"dc:identifier.uri","label":"Identifier URI","values":["https://hdl.handle.net/10919/135468"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description.abstract","label":"Abstract","values":["Auxin is a central regulator of nearly all aspects of plant growth, development, and environmental response. Despite its broad roles, the mechanisms by which a single hormone elicits such diverse and context-specific transcriptional responses remain unclear. These responses are primarily mediated by the nuclear auxin signaling pathway, which consists of ARF transcriptional activators, Aux/IAA repressors, and TIR1/AFB auxin receptors that work together to modulate gene expression. Functional diversity within these modular components, such as differences in DNA binding, protein-protein interactions, subcellular localization, and co-expression patterns is believed to act in concert to tune auxin signaling specificity. In support of our efforts to decode auxin signaling specificity, we developed an open-source, R-based analysis pipeline to process flow cytometry data generated by protoplast transient expression assays. This reproducible workflow automates transformation-based reporter analysis, streamlining quantification of gene expression and effector function in high-throughput experiments. To understand the functional diversity in ARF-mediated transcriptional responses, we performed RNA sequencing of Arabidopsis protoplasts transiently expressing irrepressible ARF variants to determine if they have distinct downstream regulatory targets. Transcriptomic analysis revealed that while each ARF appears to regulate largely overlapping sets of genes, they do so with varying efficiencies. Gene clusters preferentially regulated by specific ARFs were associated with distinct biological processes, aligning with the known developmental roles of those specific ARFs. While our transcriptomic analysis suggested that ARFs can drive distinct transcriptional outputs independently of Aux/IAAs, the extent to which specific ARF-Aux/IAA interactions contribute to this specificity remained unclear. To determine whether specific ARF-Aux/IAA interactions also contribute to transcriptional specificity, we developed a synthetic system that isolates these interactions using a recombinant protein-interaction domain from an animal ortholog. The system was validated through structural prediction, yeast two-hybrid assays, and protoplast transient expression assays. This system serves as the closest approximation to date of native ARF-Aux/IAA interactions, providing a powerful tool to dissect signaling specificity at the level of individual protein pairings. Together, the development of a streamlined data analysis pipeline, transcriptional profiling, and a synthetic interaction system provides both computational and experimental tools that advance our understanding of auxin signaling specificity. Ultimately, these insights will help explain how a single hormone can direct a vast array of developmental and environmental responses in plants."]},{"key":"dc:description.abstractgeneral","label":"General Abstract","values":["Plants produce natural chemicals called hormones to control how they grow, develop, and respond to their surroundings. One of these hormones, auxin, is particularly important as it influences almost every aspect of plant development, from the positioning of roots and leaves to environmental stress responses. Ultimately, a better understanding of auxin control over plant development could help combat global food insecurity through the development of more resilient crops tailored to a changing world. However, despite a century of auxin research, scientists still do not fully understand how this single molecule controls so many aspects of plant development. Auxin works through an internal communication network system in plant cells that relays messages through the action of specific biological machines called proteins to determine how the plant responds. To understand how these proteins communicate to facilitate different responses, we created an artificial system to study individual interactions without interference from the whole communication network. This enables us to examine how particular protein interactions control different plant behaviors. To streamline and accelerate this research, we also developed a user-friendly computer program that helps scientists quickly analyze and interpret large amounts of experimental data. We used advanced tools to identify which and how specific pieces of plant DNA, called genes, respond when a group of functionally similar proteins deliver auxin's messages. Our findings showed that these proteins communicate with essentially the same genes but yield different responses. This suggests that there are additional factors, beyond the known auxin communication network, that help determine exactly how the same message can yield distinct outcomes. Together, these studies offer new insights and practical tools for deciphering the complexities of auxin's messages. By revealing how different protein interactions guide unique responses within plants, this research helps explain how a single hormone can coordinate so many aspects of plant life. Ultimately, unscrambling auxin's code could help improve global agriculture by allowing us to produce more resilient crops in a changing world"]},{"key":"dc:description.degree","label":"Dc Description Degree","values":["Doctor of Philosophy"]},{"key":"dc:format.medium","label":"Dc Format Medium","values":["ETD"]},{"key":"dc:title","label":"Title","values":["Decoding the Transcriptional Specificity of Auxin Signaling: A Synthetic Biology Approach"]}]}],"canonical_facts":{"dc:contributor.committeechair":["Bargmann, Bastiaan"],"dc:contributor.committeemember":["Wang, Xiaofeng","Winkel, Brenda Sophia J.","Wright, Robert Clay"],"dc:contributor.department":["Horticulture"],"dc:creator":["Taylor, Joseph Sylvester"],"dc:date.accessioned":["2025-06-11T08:02:57Z"],"dc:date.available":["2025-06-11T08:02:57Z"],"dc:date.issued":["2025-06-10"],"dc:description.abstract":["Auxin is a central regulator of nearly all aspects of plant growth, development, and environmental response. Despite its broad roles, the mechanisms by which a single hormone elicits such diverse and context-specific transcriptional responses remain unclear. These responses are primarily mediated by the nuclear auxin signaling pathway, which consists of ARF transcriptional activators, Aux/IAA repressors, and TIR1/AFB auxin receptors that work together to modulate gene expression. Functional diversity within these modular components, such as differences in DNA binding, protein-protein interactions, subcellular localization, and co-expression patterns is believed to act in concert to tune auxin signaling specificity. In support of our efforts to decode auxin signaling specificity, we developed an open-source, R-based analysis pipeline to process flow cytometry data generated by protoplast transient expression assays. This reproducible workflow automates transformation-based reporter analysis, streamlining quantification of gene expression and effector function in high-throughput experiments. To understand the functional diversity in ARF-mediated transcriptional responses, we performed RNA sequencing of Arabidopsis protoplasts transiently expressing irrepressible ARF variants to determine if they have distinct downstream regulatory targets. Transcriptomic analysis revealed that while each ARF appears to regulate largely overlapping sets of genes, they do so with varying efficiencies. Gene clusters preferentially regulated by specific ARFs were associated with distinct biological processes, aligning with the known developmental roles of those specific ARFs. While our transcriptomic analysis suggested that ARFs can drive distinct transcriptional outputs independently of Aux/IAAs, the extent to which specific ARF-Aux/IAA interactions contribute to this specificity remained unclear. To determine whether specific ARF-Aux/IAA interactions also contribute to transcriptional specificity, we developed a synthetic system that isolates these interactions using a recombinant protein-interaction domain from an animal ortholog. The system was validated through structural prediction, yeast two-hybrid assays, and protoplast transient expression assays. This system serves as the closest approximation to date of native ARF-Aux/IAA interactions, providing a powerful tool to dissect signaling specificity at the level of individual protein pairings. Together, the development of a streamlined data analysis pipeline, transcriptional profiling, and a synthetic interaction system provides both computational and experimental tools that advance our understanding of auxin signaling specificity. Ultimately, these insights will help explain how a single hormone can direct a vast array of developmental and environmental responses in plants."],"dc:description.abstractgeneral":["Plants produce natural chemicals called hormones to control how they grow, develop, and respond to their surroundings. One of these hormones, auxin, is particularly important as it influences almost every aspect of plant development, from the positioning of roots and leaves to environmental stress responses. Ultimately, a better understanding of auxin control over plant development could help combat global food insecurity through the development of more resilient crops tailored to a changing world. However, despite a century of auxin research, scientists still do not fully understand how this single molecule controls so many aspects of plant development. Auxin works through an internal communication network system in plant cells that relays messages through the action of specific biological machines called proteins to determine how the plant responds. To understand how these proteins communicate to facilitate different responses, we created an artificial system to study individual interactions without interference from the whole communication network. This enables us to examine how particular protein interactions control different plant behaviors. To streamline and accelerate this research, we also developed a user-friendly computer program that helps scientists quickly analyze and interpret large amounts of experimental data. We used advanced tools to identify which and how specific pieces of plant DNA, called genes, respond when a group of functionally similar proteins deliver auxin's messages. Our findings showed that these proteins communicate with essentially the same genes but yield different responses. This suggests that there are additional factors, beyond the known auxin communication network, that help determine exactly how the same message can yield distinct outcomes. Together, these studies offer new insights and practical tools for deciphering the complexities of auxin's messages. By revealing how different protein interactions guide unique responses within plants, this research helps explain how a single hormone can coordinate so many aspects of plant life. Ultimately, unscrambling auxin's code could help improve global agriculture by allowing us to produce more resilient crops in a changing world"],"dc:description.degree":["Doctor of Philosophy"],"dc:format.medium":["ETD"],"dc:identifier.other":["vt_gsexam:44070"],"dc:identifier.uri":["https://hdl.handle.net/10919/135468"],"dc:language.iso":["en"],"dc:publisher":["Virginia Tech"],"dc:rights":["In Copyright"],"dc:rights.uri":["http://rightsstatements.org/vocab/InC/1.0/"],"dc:subject":["auxin","plant development","synthetic biology","protoplast","transient expression"],"dc:title":["Decoding the Transcriptional Specificity of Auxin Signaling: A Synthetic Biology Approach"],"dc:type":["Dissertation"],"thesis:degree_discipline":["Horticulture"],"thesis:degree_level":["doctoral"],"thesis:degree_name":["Doctor of Philosophy"],"thesis:institution_name":["Virginia Polytechnic Institute and State University"]},"updated_at":"2026-07-22T22:18:47Z"}