Accordion Content
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Inefficient methods for transformation and regeneration of recalcitrant plant species prevent widespread applications of genome editing technologies for both basic and applied research in established and emerging crop species. Overcoming these limitations is particularly relevant in monocotyledonous crops, such as maize, which alone provide most of the calories consumed by humans. In this project, maize lines expressing genes that promote regeneration, also known as morphogenic factors, will be used to provide a thorough understanding of the molecular events leading to the successful formation of new plants starting from differentiated tissue. This knowledge will be instrumental in developing new strategies for improving transformation of maize and other plant species, and will be integrated into course-based undergraduate research experiences (CUREs) as well as hands-on transformation workshops.
The proposed research will exploit a morphogenic-based system called ?GGB? to understand how certain morphogenic regulators reprogram somatic cells to develop into embryos and identify key regeneration genes that could be targeted to improve transformation efficiency in recalcitrant genetic backgrounds. This will be accomplished by the identification of direct targets of regulation of the GGB components via single-cell transcriptomic and DNA-binding approaches, and by the development of a diverse panel of maize inbred lines expressing the GGB morphogenic regulators. By exploiting the regenerative capacity of this system, protoplast regeneration, a challenging but advantageous system for the rapid generation of non-GMO edited plants, will also be revisited. This research will provide insights into the molecular basis of tissue- and genotype-dependent regeneration, helping to identify and eventually bypass roadblocks to regeneration, and will facilitate the development of high-throughput systems for genome-editing and transgenic line generation in diverse genetic backgrounds. -
The regulation of the size of meristems, groups of plant stem cells, plays an important role in plant development and crop productivity. Increases in meristem size achieved during the domestication of several crop species resulted in bigger fruits and inflorescences, and continue to offer great potential to increase yield. This proposal will provide a thorough understanding of the genetic and molecular mechanisms essential for increasing ear size in maize, a major crop worldwide, that can potentially translate to increased yields in commercial hybrids. The research will be integrated with active scientific training of high school students, and with a new research-based teaching module that combines classic genetic analysis with translational research tailored to graduate students.
At the heart of the regulatory network controlling meristem size is the transcription factor WUSCHEL (WUS). WUSCHEL function has been proposed to have diversified between monocot and eudicot species. In maize, however, WUS function has yet to be explored, despite its importance in plant development and its recent use in plant transformation technologies. The proposed research will reveal the function of duplicated WUS genes in maize and their role as transcriptional repressors by combining genetic analysis and transgenic approaches with single cell transcriptomics of maize inflorescences. This work aims to uncover key mechanistic details of meristem size regulation in maize and monocots in general, answering long-standing questions regarding evolutionary conservation or diversification of WUS function, and to reveal new regulatory targets that could be genetically manipulated to increase maize yield and improve transformation efficiency.
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Transcription factors are proteins that bind to short DNA sequence motifs in regulatory regions of their target genes and thus control the gene expression changes responsible for plant developmental programs and environmental responses. In crop species, variation in transcription factors and the regulatory regions they bind have been frequent drivers of productivity gains during domestication and modern breeding, and continue to offer great potential for further trait engineering. Yet, in plant genomes, the vast majority of transcription factor-DNA binding events and the gene expression changes they elicit remain largely uncharacterized, restricting the development of new varieties that meet the challenges of modern agriculture. In this project, detailed regulatory information maps and new methodologies will be generated to identify relationships between transcription factor binding and variability in gene expression, providing new tools for the rational design of crops with improved traits. These methods have the potential to fundamentally transform crop improvement strategies to adequately feed the expanding global population. To extend the reach of the project, scientists working under this award will provide specialized training in genomics and bioinformatics to students with diverse backgrounds.
A large portion of plant genetic variation is regulatory and resides in non-coding regions, spaces that are often vast in genomes such as maize. Mining functional elements from these spaces represents a major challenge, in part because while potential transcription factor (TF) binding sites are naturally abundant within a genome, only a small fraction is actually bound and able to affect expression. Empirically cataloging plant TF binding events and their contribution to transcriptional outputs is therefore a priority for understanding transcriptional networks and trait variation. This project will develop TF-DNA interaction methods that enable comparative analysis of multiple genetic backgrounds, resulting in the generation of high-resolution maps of conserved regulatory regions and accession-specific variants that can be linked to transcriptional programs. This approach will be applied in two species with different genomic properties: maize, a major monocot crop with a large genome; and Arabidopsis, a model eudicot with a compact genome. Because many TFs do not function in isolation but instead interact with other proteins that can alter their DNA binding activity, this project will also develop techniques to better understand the contribution of TF pairs to transcriptional regulation. To directly link TF binding events to phenotypic outcomes, specific regulatory elements in key genes controlling plant architecture and reproductive development will be functionally characterized through precise genome editing. Such experiments will demonstrate how modulation of regulatory regions can be used to create subtle changes in gene expression levels or spatial expression patterns that may result in advantageous phenotypes.