The plant growth hormone auxin is a small molecule with an amazing diversity of functions, from the development of shoots and roots to specific responses to biotic and abiotic cues. How is this possible? The nuclear auxin signaling pathway itself is short and involves three specific components: a receptor (TIR/AFB) which is the F box component of an E3 ubiquitin ligase, a transcription factor (AUXIN RESPONSE FACTOR, ARF), and a repressor (Aux/IAA). High affinity binding of auxin by the receptor and repressor, leads to ubiquitination and degradation of the repressor, freeing the ARF transcription factor (TF) to bind cis-regulatory elements (AuxREs) in target genes. With this simple mechanism, how does auxin provoke such a diversity of responses? Part of the answer comes from the fact that each component belongs to a gene family. In maize, there are 8 receptors (TIR/AFBs), 33 repressors (Aux/IAAs) and 38 TFs (ARFs), allowing for many possible combinations. Recent studies have highlighted the importance of combinatorial interaction of auxin signaling components in determining auxin affinity, rates of protein turnover, DNA binding profiles, and rates of transcriptional activation. To dissect the specificity of the developmental response, it is critical to know the spatiotemporal relationships of the signaling components. We propose to dissect the gene regulatory network connecting auxin perception with development of specific regions or domains within the maize inflorescence.
We hypothesize that each developmental domain in maize is specified by a distinct auxin signaling module. Connecting the molecular network with tissue-level events will require a detailed understanding of which interactions occur in specific tissues and at specific times, how the components diverge, and what the target genes are. This challenge will be met with a highly integrated approach, incorporating genomic and synthetic biology tools with sophisticated informatics and data visualization techniques. Our approach will answer critical developmental questions in a crop system, provide genome-level understanding of how molecular signaling pathways diverge to evolve new functionalities in different contexts, and will have widespread implications for many biological processes and all plant species.
The maize auxin gene catalog
Auxin-related genes are highly conserved in plants. However, given the vast developmental diversity among plant species, many are likely to display varying degrees of functionality with important consequences for crop fitness and yield. To investigate the potential for such differences, we are creating a catalog of manually annotated maize auxin-related genes for functional analysis. Our labs primarily study the reproductive structures of maize. Therefore the following gene annotation and expression analysis focuses predominantly on genes expressed in tassel and ear.
Developmental dynamics of auxin gene expression
We are performing extensive RNA-seq analysis of auxin mutants including vt2, bif2, Bif1/Bif4 and ba1 compared to B73. The RNA was extracted from the top 1mm (early stage, IM/SPM) and the bottom 1mm (later stage, SPM/SM) of 3-4mm tassels dissected from 3-4 week old plants grown in the field. This allows us to identify genes differentially expressed in all three functional domains (axillary meristem, bract and boundary) in the inflorescence.
We are using a newly developed genomic approach to identify the downstream targets of ZmARF binding. Thanks to the DAP-seq technique, we are describing the binding properties and behavior of phylogenetically distinct ARFs as well as identifying many unknown auxin regulated genes. Since auxin regulates many developmental processes and pathways, these datasets represent an important resource for the community.
Synthetic Biology: functional analysis using auxin response circuits
Connecting auxin signaling modules with tissue-level events will require a detailed understanding of which interactions occur in specific tissues and at specific times. One way we are approaching functional analysis of the auxin signaling interactome is through a synthetic auxin signaling system in yeast. This systems enables co-expression of user-defined auxin signaling modules: receptors, repressors, transcription factors, and transcription factor binding sites. Auxin-induced signaling dynamics can then be measured for each module, providing information on sensitivity to auxin levels and the speed and magnitude of the auxin response.
Our goal is to use the tremendous genetic tools and resources available in maize to identify new genes that regulate auxin function and establish functional domains within maize inflorescences. This will be accomplished by a combination of traditional genetics, genome editing and mutagenesis approaches.
We propose to build multi-level interaction networks from auxin input to developmental output for specific functional domains in maize. Auxin related regulatory and information passing mechanisms in maize forms complex gene relationships diversely underlie genomics, transcriptome, metabolomics and proteomics data. Interaction network, a powerful mathematics model, could integrate these relationships from multiple biological levels and biological processes into one dynamic complex network. This model could investigate, demonstrate the auxin signaling, and could use for predict and hypothesis testing on stimulating and mutation of specific behaviors of auxin.
Training and Development
Our research team is committed to mentoring a diverse group of future scientists and providing purposeful first contact moments with research. This project is training high school, undergraduate and graduate students, and postdocs in interdisciplinary genomics and synthetic biology research. Team members are implementing research-based undergraduate courses that directly engage students in experiments addressing key questions in our research project. We are also developing new K-12 and general public outreach activities with a focus on maize development and domestication. This project is of benefit to society through training of participants, K-12 and public outreach, and integration of research and undergraduate education.
Any opinions, findings, and conclusions or recommendations expressed in this material are those of the author(s) and do not necessarily reflect the views of the National Science Foundation.