Heterosis, or hybrid vigor, refers to the phenomenon in which F1 offspring of two genetically distinct parental lines exhibit phenotypes outside the range of its parents. Agricultural breeding programs commonly utilize heterosis to enhance yield, disease resistance, and other desirable traits in both crops and livestock, despite the labor-intensive field tests required for hybrid breeding programs. Despite its importance, the mechanisms by which genetic variation between two parents combine to produce non-additive phenotypes in hybrids remain unclear. To identify molecular components that may contribute to heterosis, we analyzed paired proteomic and transcriptomic data from leaf tissue of maize hybrids and their inbred parents. Expression levels of plastid proteins involved in translation and photosynthesis were increased in seedling leaf tissue of hybrids relative to mid-parent and were positively correlated with heterosis levels in adult plants. Conversely, levels of proteins involved in stress responses were reduced in seedling leaf tissue of hybrids relative to mid-parent and were negatively correlated with heterosis levels in adult plants. An ethylene biosynthesis mutant copied the hybrid proteome, indicating that the most of the altered protein levels in hybrids are downstream of the reduction in ethylene biosynthesis. Protein expression patterns across the developmental gradient of hybrid leaves were altered in comparison to the inbred parents. Proteins involved in photosynthesis and translation were reduced in the basal zone of the leaf but increased in the mature zone. This suggests that hybrids have more precisely controlled expression patterns across the developmental gradient which could contribute to their greater fitness. The same expression patterns were observed in the ethylene mutant, indicating that reduced ethylene biosynthesis largely mediates the developmental differences between hybrids and inbreds.

Developmental and Hormonal Regulation of Heterosis in Maize: A Spatial Multi-omic Dissection

Heterosis, or hybrid vigor, is a fundamental yet poorly understood biologicalphenomenon in which F₁ hybrids exhibit superior phenotypic performance compared to their inbred parents. Despite its widespread use in agriculture—particularly in many crop breeding programs—its molecular basis remains elusive. This dissertation investigates the developmental and hormonal regulation of heterosis through a multi-omic analysis of maize seedling leaves, integrating transcriptomic and proteomic data across spatial and genotypic contexts.

In Chapter 1, paired transcriptome and proteome datasets from 15 maize hybridsexhibiting a range of plant height heterosis were analyzed using weighted gene co-expression network analysis (WGCNA). Independent co-expression networks for RNA and protein expression revealed low overlap in network topology but strong functional convergence. Modules most strongly correlated with heterosis were enriched in pathways associated with plastid proteostasis, ribosome biogenesis, and tetrapyrrole synthesis. A small subset of hub genes—such as plastid ribosomal proteins and folding chaperones—emerged as central regulators across omic layers, highlighting the role of translation and proteostasis stress suppression in hybrid performance.

Chapter 2 expands this investigation by incorporating spatial profiling of leafdevelopment and hormonal perturbation using the ethylene-deficient acs2/6 mutant. Proteomic and transcriptomic analyses across four consecutive sections of V3 leaves in maize demonstrated that heterosis-associated molecular phenotypes are developmentally specific. Core subunit of photosynthesis-associated protein complexes showed overdominant expression in mature leaf regions (source), while TOR signaling components were elevated at the base (sink). Ethylene repression alone partially recapitulated hybrid-like proteomic dynamics but failed to induce the widespread transcriptional reprogramming observed in the hybrid. The hybrid uniquely displayed repression of chromatin remodeling complexes and activation of translation machinery, indicating large-scale transcriptional reprogramming as a key feature of hybrid vigor.

Collectively, the results support a multi-tiered model in which heterosis is driven bymolecular complementation of photosynthetic complexes, sugar-activated TOR signaling, defense hormone repression and hybrid-specific chromatin remodeling. This spatially coordinated reprogramming enhances growth potential and translational capacity while modulating defense and stress responses. The findings offer new mechanistic insights into heterosis and suggest biomarkers and regulatory modules that may inform new breeding strategies.

Heterosis is a crucial facet of evolution that produces higher quality, healthier, and more resistant offspring relative to parents. Conversely, inbreeding depression is the lack of vigor because of overexpressing harmful or detrimental traits. Heterosis is a fairly unexplored field in biology, and much is still unknown regarding the effects of heterosis on the maize embryo and its implications for many metabolic pathways that occur there. In Vivo research on the proteomic effects of heterosis in Zea mays (maize) embryos will contribute to an increased knowledge of heterosis in Maize embryos. Improved heterosis of maize can lead to increased food production, more extensive livestock health, and fuel ethanol production. My thesis sought to identify which protein levels in the maize embryo are elevated or decreased and the potential role of these proteins in heterosis. We identified that levels of histone linker 1 (H1) and several enzymes involved with lipid biosynthesis are elevated in the hybrid. This finding suggests that hybrids may experience reduced stress by lowering the expression levels of transposable elements. Furthermore, levels of glutathione S-transferase and hsp60 proteins were reduced in the hybrid embryo, indicating lower levels of metabolic stress. By identifying protein groups and individual proteins that were not expressed at midparent levels, we identified metabolic pathways that are affected by heterosis, which contributes to a deeper understanding of the mechanism of heterosis in maize embryos.

Study of the regulation and location of the protein products of genes is essential for understanding the phenotype of the organism. The quantitative and qualitative control of protein production, as well as post-translational modification and subcellular localization of the protein, in part determine the effect of a gene on a biological system. As it becomes more apparent that complex traits are controlled by effects from many loci, it has become more imperative that we seek a proteome-wide understanding of protein regulation and localization. The proteomes of four maize subcellular organelles were characterized by comparison to their source tissues, defining both organelle-enriched and depleted protein sets. High confidence localizations to organelle were obtained for plasma membranes (2154), mitochondria (1079), glyoxysomes (461), and plastids (539). Many cases of localization were novel or revised existing annotations, including that of nearly 40% of localized maize classical genes. Many proteins localized to multiple compartments, including a large overlap between mitochondrial and chloroplast proteins, whereas few proteins were shared between the chloroplast and non-mitochondrial organelles. A machine learning approach was used to identify the expressible gene sets of sorghum and gene set annotations from versions 2 and 4 of the maize genome. These gene sets were identified by a classifier trained using only DNA methylation data as model features. Synteny was leveraged to identify species-specific expressible genes, revealing enrichment of biotic and abiotic stress-associated genes in the species-specific pools. Expressible gene sets also provide evidence for express-ability of gene models absent from the maize version 2 annotated filtered gene set or the maize version 4 annotated gene set.

Maize is a major staple food crop with important agricultural and agronomic impact worldwide. However, fungi of the genus Fusarium can cause diseases in maize, resulting in significant economic losses. Immunity requires both transcriptional and translational regulation for an effective defense response. To investigate how fungal challenge affects maize, we generated RNA-Seq and proteomics data from plants that had been challenged with heat-killed Fusarium venenatum hyphae over a ten-point time course. Our findings indicated that maize can exhibit different transcriptional and translational responses to fungal elicitation. Weighted gene co-expression network analysis (WGCNA) resulted in identification of a module in which RNA does not change over time and protein abundance increases, providing examples of translational re-programming in maize. GO analysis on the genes in this module revealed enrichment for protein modifications and vesicle-mediated transport. Performing WGCNA on the fold change rank orders yielded four main patterns of RNA and protein expression. In the largest rank-order module, RNA and protein abundance both decreased over time; this module was most enriched for genes involved in plant development, providing evidence for the shift from growth towards defense. The next three largest modules exhibited increasing protein abundance, and were enriched for genes involved in ubiquitination and responses to stimuli, which are all involved in defense. Surprisingly, about 15% of detected genes are in Module 2 and tend to have decreasing RNA expression and increasing protein abundance. Transcriptional and proteomic analyses will promote greater understanding of the complex regulation of the plant response to pathogen challenge.

Veterans returning from deployment in the Middle East have displayed chronic asthmatic-like symptoms after exposure to burn pits and sandstorms. These soldiers also encounter stressful environments during deployment, introducing another factor that could alter their immune response. In this thesis, we have investigated the impact of acute stress on lung inflammation in murine models, focusing on the role of innate lymphoid cells (ILCs) during exposure to the allergen Alternaria alternata (Alt) and burn pit constituents (BPCs) which include dioxins, hydrocarbons, and particulate matter. Our previous studies have shown that combinatory exposure to Alt and BPCs induces a mixed type 2 and non-type 2 inflammatory response, characterized by increased neutrophilia and a shift from ILC2 to ILC1-like populations. Single-cell RNA sequencing (scRNA-seq) of lung samples showed upregulation of neutrophilia-associated genes and stress-related genes in ILCs from Alt+BPC-challenged mice, indicating that stress exacerbates inflammatory responses. Here, we present a novel model to reflect an acute stress event followed by inhalation of BPCs or Alt focusing on analysis of lung granulocytes and innate lymphoid cells. Results indicated a significant decrease in different ILC subpopulations of stressed mice after either allergen or BPC exposure. This decline reflects reduced neutrophilia in non-type 2 inflammation and trending decreases in eosinophilia in type 2 inflammation. This study concludes that acute stress alters the immune response to lung inflammation. These findings suggest that stress, combined with BPC exposure, might contribute to the respiratory issues observed in exposed military personnel.