| NSF Org: |
IOS Division Of Integrative Organismal Systems |
| Recipient: |
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| Initial Amendment Date: | September 8, 2016 |
| Latest Amendment Date: | July 22, 2021 |
| Award Number: | 1546873 |
| Award Instrument: | Continuing Grant |
| Program Manager: |
Gerald Schoenknecht
IOS Division Of Integrative Organismal Systems BIO Directorate for Biological Sciences |
| Start Date: | September 15, 2016 |
| End Date: | June 30, 2023 (Estimated) |
| Total Intended Award Amount: | $3,643,117.00 |
| Total Awarded Amount to Date: | $3,843,077.00 |
| Funds Obligated to Date: |
FY 2017 = $763,839.00 FY 2018 = $785,897.00 FY 2019 = $47,774.00 FY 2020 = $713,104.00 FY 2021 = $136,146.00 |
| History of Investigator: |
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| Recipient Sponsored Research Office: |
121 UNIVERSITY HALL COLUMBIA MO US 65211-3020 (573)882-7560 |
| Sponsor Congressional District: |
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| Primary Place of Performance: |
301 Bond Life Sciences Center Columbia MO US 65211-1230 |
| Primary Place of
Performance Congressional District: |
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| Unique Entity Identifier (UEI): |
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| Parent UEI: |
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| NSF Program(s): |
Plant Genome Research Project, Cross-BIO Activities |
| Primary Program Source: |
01001718DB NSF RESEARCH & RELATED ACTIVIT 01002122DB NSF RESEARCH & RELATED ACTIVIT 01001617DB NSF RESEARCH & RELATED ACTIVIT 01002021DB NSF RESEARCH & RELATED ACTIVIT 01001819DB NSF RESEARCH & RELATED ACTIVIT |
| Program Reference Code(s): |
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| Program Element Code(s): |
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| Award Agency Code: | 4900 |
| Fund Agency Code: | 4900 |
| Assistance Listing Number(s): | 47.074 |
ABSTRACT
Imagine any characteristic of plants - from development of shoots and roots to responses to the environment and pests - and it is almost certainly shaped by the plant growth hormone, auxin. Plant cells have the ability to detect and respond to auxin. Thus, characterizing the network of molecular components that involved in detection and response will improve our understanding of the multiple auxin effects. The primary goal of this project is to identify the auxin-regulated molecular networks in corn, an economically important crop in USA. Characterizing the roles these networks play in the growth and development in the tassel and ear of corn will help devise strategies to improve yields. A team of scientists from four different American universities will carry out this work using the most modern biological techniques. This work will fundamentally change the landscape of developmental biology in corn, and will significantly accelerate the transfer of knowledge from the lab to the field. Results from this work will made publically available. The research team is strongly committed to mentoring a diverse group of future scientists and to increasing broad scientific literacy. This project will organize workshops aimed at expanding the professional skillsets of undergraduate, graduate and postdoctoral trainees. Scientists from the four institutions will collaborate to develop training modules that will involve hands-on experimentation. These modules will specifically target audiences from K-12 schools, colleges, and the public at large.
This project addresses a cornerstone of developmental biology: how auxin action is specified by context-specific deployment of signaling components, and in particular, how auxin controls reproductive organogenesis. Team members have amassed a wealth of genetic, genomic and molecular resources in maize to answer the question of how axillary meristems are initiated to give rise to the reproductive structures in the tassel and ear. Prior work has shown that components of the auxin signaling machinery are expressed in three functional domains within the inflorescence: the suppressed bract leaf, the boundary domain, and the axillary meristem. Connecting auxin signaling modules with tissue-level events will require a detailed understanding of which interactions occur in specific tissues and at specific times. This challenge will be met with a highly integrated approach incorporating genomic and synthetic biology tools with sophisticated informatics and data visualization. Specifically, this project will: 1) define and characterize the auxin signaling modules (receptors, repressors, transcription factors) operating within functional domains of the inflorescence; and 2) construct gene regulatory networks incorporating co-expressed genes and targets of the auxin signaling modules.
PUBLICATIONS PRODUCED AS A RESULT OF THIS RESEARCH
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PROJECT OUTCOMES REPORT
Disclaimer
This Project Outcomes Report for the General Public is displayed verbatim as submitted by the Principal Investigator (PI) for this award. Any opinions, findings, and conclusions or recommendations expressed in this Report are those of the PI and do not necessarily reflect the views of the National Science Foundation; NSF has not approved or endorsed its content.
Almost every aspect of plant development and interaction with the environment, is regulated by the plant growth hormone, auxin. To understand how auxin regulates such diverse processes, it is important to characterize the network of molecular components that enable the plant to detect and respond to auxin. The primary goal of this research was to define these auxin-regulated molecular networks regulating the development of reproductive structures (tassel and ear) of the economically important crop plant, maize (corn).
Intellectual Merit
Our first objective was to identify and characterize auxin signaling genes functioning in specific domains within the maize inflorescence by analyzing the expression of three gene families at the core of auxin signaling by RNA in situ hybridization and by determining function by reverse genetics and yeast synthetic biology. We obtained a collection of transposon insertion mutants in 10 Zea mays AUXIN RESPONSE FACTORS (ZmARFs), 7 Zea mays AUXIN SIGNALING F-BOX (AFB) receptors (ZmAFBs) and CRISPR-Cas9 edited additional genes. Unfortunately, single and double mutant combinations did not display obvious developmental defects in inflorescences. This was likely due to the high degree of redundancy, which was also revealed by the overlapping expression patterns of the genes. Therefore, we functionally annotated the core auxin signaling components that were expressed in inflorescences, by recapitulating the complete nuclear Auxin Response Circuit in yeast.
All 16 auxin/indole-3-acetic acid (ZmAux/IAA) repressor proteins were degraded in yeast in response to auxin with rates that depended on both the auxin receptor and repressor identities. When fused to the RAMOSA1 ENHANCER LOCUS2 (REL2) corepressor, ZmAux/IAAs were able to repress ZmARF transcriptional activity. The ZmAFB2/3 b1 auxin receptor, was more sensitive than the Arabidopsis ortholog and allowed for rapid circuit activation upon auxin addition. In addition, we compared the activation profiles of Arabidopsis and maize ARFs on standardized synthetic promoter variants in yeast, and found that specificity in auxin responses cannot be explained by differences in promoter preferences between ARFs.
Once repressor activity of ZmAUX/IAAs had been analyzed in yeast, we performed testing of several variants of one repressor, BARREN INFLORESCENCE2 (BIF4), with altered stability, in stably transformed maize lines. The goal was to engineer maize plants with altered inflorescence architecture by modulating auxin signaling in inflorescences. Events expressing all three constructs were isolated. Unfortunately, we could not detect consistent effects on inflorescence architecture, despite detecting clear nuclear expression of each fusion protein by confocal microscopy. Attempts to complement Bif4 with the wild type version of the BIF4 gene (pBIF4::VENUS:BIF4) failed, indicating that either the expression levels of the transgenes were too low to impact development or that the fusion protein was non-functional.
To further expand the synthetic biology toolbox in maize, we successfully built and tested a prototype serine-integrase gene expression recorder in maize protoplasts, which is currently being stably transformed into maize to build a two-integrase order-of-expression tracker.
Our second objective was to build gene regulatory networks to identify additional components functioning in specific domains of the maize inflorescence using multi-omic data integration and forward genetics. DAPseq was performed with 14 ZmARFs and RNAseq was performed with developing tassel meristems of 7 auxin mutants compared to wildtype and gene regulatory network analysis performed. Multiomic data integration software was developed as well as a deep learning method. In addition, genetic analysis was used to identify and characterize the function of 5 new genes functioning in inflorescence development (barrenstalk2, lateral suppressor1, tassel-less4, barren inflorescence3, and needle1). Enhancer screens were performed which, although they did not identify true enhancers, did lead to the isolation of new inflorescence mutants which are currently being mapped and characterized.
Product outcomes include 4 PhD theses, 9 undergraduate honors theses, 25 research articles, 8 review articles and 3 methods papers, so far, as well as new multi-omic integration software.
Broader Impacts
Training was provided to 8 postdoctoral scholars, 3 technical staff, 12 PhD students, 2 postbaccalaureate students on a REPS supplement, 27 undergraduate students including 8 Freshman Research in Plant Sciences (FRIPS) students and 6 REU students on intercampus exchange. In addition, 74 Whitman College undergraduate students participated in projects related to objective 1 through development of a CURE (course based undergraduate research experience). In addition to training in interdisciplinary research, quarterly professional development/skills workshops were held with all members of the project team. Moreover, a bioinformatics training workshop and a career panel were hosted by the team. We developed educational outreach materials for K-12 and the general public, which were utilized at various venues. We developed the KBCommons framework for maize which has been further expanded by addition of the “3DOmics Studio Tool” for supporting interactive functional and pathway enrichment analysis and “Comparative and Cross-species Multiomics Translation” (CCMT) Tool to support multi-omics data integration.
Last Modified: 12/20/2023
Modified by: Paula C Mcsteen
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