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Plasmodesmata are nanoscale, membrane lined pores that span the cell wall of neighbouring plant cells. The presence of these structures enables cell-cell movement of substances important for plant growth and development. This thesis describes the nature of plasmodesmata and explores their importance at various biological scales within the model plant Arabidopsis thaliana. The first two experimental chapters focus on selected molecular components of individual plasmodesmata. I show that sphingolipids, a lipid class enriched in the membranes of these structures, have a functional role in shaping the pores. Sphingolipid alterations resulting from mutation in the Phloem Unloading Mediator gene or in the Long Chain Base Kinase 1 gene result in plasmodesmata morphological changes with consequences on cell-cell permeability. Moving to the protein component of plasmodesmata, I highlight that the family of Multiple C2 and Transmembrane Region Proteins influences long distance movement in the phloem, a vascular conduit in plants. I also show that loss of selected members of the family causes severe developmental phenotypes. Additionally, my results suggest potential interactions between sphingolipids and these proteins. The following experimental chapters look at increasingly larger biological scales. Employing serial block electron microscopy, I develop computational tools to study the distribution and density of plasmodesmata over entire cellular interfaces, and the thickness of the wall they span. The proof of concept examples interestingly show differences in spatial patterns, depending on the interface, and suggest an influence of sphingolipids on cell wall thickness. I move to the tissue scale, focusing on movement in the phloem, and identify EMS-Mutagenized BRI1 Suppressor 3 as a suppressor of restricted phloem unloading in lines that over-accumulate callose, a polysaccharide occluding plasmodesmata. The protein might specifically control correct folding of callose synthase enzymes within the endoplasmic reticulum. Lastly, looking at the whole plant level, I show that induced closure of plasmodesmata within a specific phloem vascular domain, that of Sister of APL, can have reproducible impacts on the rate of growth of axillary buds into branches. The effect is observed in a range of genetic backgrounds and might involve Pin-Formed proteins.

Low nutrition availability in the soil can be a major limitation of plant growth. To improve nutrient acquisition, the majority of land plants engage in symbiosis with arbuscular mycorrhizal (AM) fungi. The accommodation of fungal colonisation structures in the roots requires their radical reprogramming. This starts during pre-symbiotic communication, where signals are exchanged between the fungus and plant across the rhizosphere. The receptor D14-LIKE emerged as a vital component of this pre-symbiotic communication when it was found to be absolutely required for symbiosis in rice. However, the broader relevance of the receptor, both in terms of functional conservation across plant species and its relation to other pre-symbiotic plant signalling components, remained unclear. The aim of this thesis was to elucidate these two key points. To address the fragmented picture of fungal signals, plant receptors and signalling pathways, a large scale transcriptomic experiment in rice was conducted to tie D14L together with other distinct pre-symbiotic components. In the absence of D14L-mediated signalling, rice was found to be compromised in the perception of germinated spore exudates, as well as specific chitinaceous signals, meaning that normal transcriptional reprogramming could not be achieved in response to any of these treatments. In addition, the functional conservation of D14L signalling was explored using trans-species complementation experiments. It was found that the Arabidopsis homolog AtKAI2 could complement the developmental phenotype of the d14l rice mutant, but not symbiosis. Likewise, D14La from early diverging Marchantia polymorpha and Marachantia paleacea could rescue developmental phenotypes in d14l rice, but again failed to complement symbiosis. This demonstrated a functional separation between developmental and symbiotic signalling. The data generated during my PhD foster D14L as a central node for multiple inputs to pre-symbiotic reprogramming, and provides new insights into pre-symbiotic communication mechanisms which are required for the successful establishment of symbiosis.

Arabinogalactan proteins (AGPs) are proteoglycans heavily substituted by arabinogalactan polysaccharides. These are composed of arabinose and galactose, and minor sugars such as glucuronic acid (GlcA), fucose and xylose. The arabinogalactan polysaccharides do not decorate classical AGPs exclusively, but they can also be found decorating a wide range of proteins. Arabinogalactan proteins have been implicated in many processes of plant development. Recently, AGPs were proposed to bind and store calcium at the plasma membrane. They are extracellular, and are localised mainly at the plasma membrane via a GPI-anchor. They can also be soluble in the apoplast. Their low abundance, chemical similarity and high functional redundancy have hindered their study. My strategy to overcome these difficulties was to study knock-out Arabidopsis thaliana plants of glycosyltransferases that transfer sugars specifically onto AG-polysaccharides. Glucuronic acid makes up about 10% of the arabinogalactan polysaccharide structure in Arabidopsis thaliana cell culture AGPs. Previously, the glucuronic acid transferase A TGLCA T14A, a member of the CAZy Glycosyl Transferase 14 family, was shown to transfer GlcA specifically onto AGPs, and knock-out Arabidopsis plants showed a 30% reduction in [Me]GlcA substitution in AGP-enriched preparations. However, no clear growth phenotype was observed. The characterisation of knock-out plants of other GT14 family members and combinations thereof is described here. Based on previous studies (Lamport and Várnai, 2013), I assayed in vitro the calcium binding capacity of AGP extracts from WT and knock-out plants. The results showed that AGP extracts from knock-out plants can hold less calcium than WT plants in vitro. A wide range of plant growth phenotypes were identified. Growth phenotypes can be explained by changes in the cytoskeleton and deficiencies in calcium signaling. Our evidence suggests links between structural deficiencies of extracellular proteoglycans to extracellular calcium and cytoskeleton. This research has the potential to create a new model system for the study of molecular mechanisms dependent on calcium that drive cell expansion, division and differentiation in plants.

The symbiosis between plants of the legume family and nitrogen-fixing rhizobia underpins global food security. Legume crops are a major source of protein in human diets, either directly or indirectly as feed for livestock. Application of inoculant rhizobial strains is common practice in many areas, as plant growth is often nitrogen limited and the symbiosis can significantly enhance yields. However, rhizobial strains and outcomes of the symbiosis vary widely. This variation has also been studied by evolutionary biologists interested in the stability of mutualisms. They proposed that plants may prevent establishing symbioses with ineffective strains (partner choice), or provide them with fewer resources (sanctioning). I studied both mechanisms, combining modelling and experimental approaches. Mathematical modelling was used to predict how plants should allocate resources to maximise growth rates, depending on rhizobial nitrogen provision and carbon requirements and on soil nitrogen conditions. The use of marked mutant strains – easily distinguishable and differing in a single rhizobial characteristic – overcame previous experimental difficulties. It was found that pea (Pisum sativum L.) plants are not able to exert partner choice, but do sanction in a more complex way than was previously established. In line with model predictions, resources were preferentially allocated to the single – best available – strain, so that resources allocated to an intermediate-fixing strain depended on whether or not a strain providing more nitrogen was available. Contrary to model predictions, there was no indication of discrimination based on rhizobial carbon requirements. The results cannot be explained by resource allocation in proportion to nitrogen received, and indicate systemic integration of information from different nodules. I formulate a hypothesis about the underlying plant regulatory mechanisms, and discuss implications of the results for selecting inoculant strains and enhancing yields in the field. Future work will rely on further integration of theoretical and applied methods and perspectives.

Hybridisation is a common evolutionary process that can arise in primary or secondary contact. Gene ow and/or reproductive isolation between hybridising taxa can be explored in hybrid zones. Therefore, a (homoploid) hybrid zone in north-west Spain between Hyacinthoides non-scripta and H. hispanica was studied. The centre occurs west to east across the Galicio-Duero Mountains with H. non-scripta distributed north, and H. hispanica south of the centre. The hybrids' genome sizes and phenotypes represented a range of intermediate states between their parents. Crossing and seed germination experiments revealed a low inter-speci c barrier, and the hybrids showed similarly good tness. Genome wide markers for large genome species were designed from transcriptomes. Diagnostic SNPs between H. non-scripta and H. hispanica were targeted and re-sequenced with multiplexing PCR. Coalescence analyses suggested a Pleistocene origin of parapatric speciation between H. non-scripta and H. hispanica. These results are supported by shared inter-speci c polymorphisms, the lack of recent hybrid generations and of parental individuals in sympatry. Di erential introgression patterns between the organellar and nuclear genomes revealed that formerly H. hispanica ranged further north but was swamped by H. non-scripta alleles. Asymmetric hybridisation was reasoned by absence of backcrosses between northern hybrids to H. non-scripta, but presence between southern hybrids and H. hispanica. Combining these results, a southwards movement of the hybrid zone centre caused by climate change (and adaptive introgression), or inter-speci c di erences in owering time was suggested. Cline patterns revealed cyto-nuclear incompatibilities, which could evolve through divergent adaptation of the organelle to climate and a delayed selection on nuclear inter-acting loci. Both species are in secondary contact in the UK due to recent introduction(s) of H. hispanica and garden variants, which is considered to cause genetic pollution of native H. non-scripta. Therefore, a conservation study is in progress, in which this diagnostic marker system for bluebells is applied.

The colonization of the arid continental surface by plants was one of the milestones in Earth's history. Morphological innovations, such as the origin of complex 3D tissues, allowed the successful colonization and radiation of plants on land. The epidermis is the outermost plant tissue that constitutes the interface between the plant and the environment. Thus, the evolution of epidermal cells was crucial for the adaptation of plants on the terrestrial arid environment. I undertook a combined approach that aims to understand the evolutionary trends that drove land plant colonization and the genetic mechanisms that underlie the development of the epidermis. This approach includes: 1) analyses of plant transcription factors (TFs) families distribution and diversification, with a particular focus on the basic Helix-Loop-Helix (bHLH) TF family, and 2) functional characterization of a putatively conserved bHLH TF subfamily involved in epidermal cell development in land plants. Here, I showed that there was a stepwise increase in the number of transcription factor (TF) families and bHLH subfamilies that predated the colonization of the terrestrial surface by plants. The subsequent increase in TF number on land was through duplication within pre-existing TF families and subfamilies. Moreover, a similar trend occurred in metazoan bHLH TF, suggesting that the majority of innovation in plant and metazoan TF families occurred in the Precambrian before the Phanerozoic radiation of land plants and metazoans. Furthermore, I demonstrated that the function of IIIf bHLH TFs in controlling the development of the epidermal cell layer is conserved between liverworts and angiosperms. This suggests that IIIf bHLH TFs are ancient and conserved regulators of epidermal cell development since the early colonization of the land by plants. Moreover, these bHLH TFs were recruited during the evolution of land plants to control the development of seemingly unrelated morphological characters in specific lineages of extant land plants. The recruitment of ancient developmental regulators to control distinct and unrelated developmental processes in land plants might underlie the huge morphological and taxonomic radiation of plants on land.

Plants encounter various biotic and abiotic stresses daily and have developed defense mechanisms to overcome these challenges. One key system involved in these defense mechanisms is the ubiquitin (Ub)-26S proteasome system (UPS), which targets malfunctioning proteins for degradation through Ub-tagged proteasomal pathways. The E3 ligases, specifically S-phase kinase-associated protein 1 (SKP1), Cullin 1 (CUL1), and F-box (SCF) complexes, play crucial roles in this process by recognizing and tagging specific protein substrates. Arabidopsis thaliana, with over 700 F-box proteins, has the largest group of E3 ligases, yet only 5% have been functionally characterized. Phylogenetic relationships among 111 plant species have identified four clusters of F-box genes, including a cluster with more conserved F-boxes, referred to as core Arabidopsis F-box (CAF) genes. Given that CAF genes have more known functions compared to other clusters, this dissertation hypothesizes significant potential for discovering new functions among the uncharacterized F-boxes within this group.Considering the evolutionary conservation of most CAFs, I adopted a genetic approach to investigate the roles of CAFs during seed germination and seed development. To address the challenges posed by functional redundancy of duplicated CAF genes and the lethality associated with constitutive F-box overexpression in transgenic plants, I created a library of inducible overexpression lines for 40 CAF genes, many of which lacked known biological functions. By systematically examining the effects of conditional overexpression of these 40 CAFs, I found that CAF overexpression during seed germination and seed development can positively or negatively regulate radicle rupture growth, thus controlling the germination process. Specifically, I identified 24 CAFs that enhance radicle rupture and two that inhibited it by interfering with abscisic acid (ABA)-mediated germination suppression. Induction of CAFs during seed developmental stages successfully enhanced germination under ABA treatment, with five genes enhancing and 12 inhibiting radicle rupture, indicating that CAFs play a development-dependent role in seed germination. Overall, I successfully transferred 40 CAFs into plants using an inducible overexpression method and categorized their impact on seed germination and seed development under ABA treatment.Next, I focused on studying the F-box substrate MULTIPLE ORGANELLAR RNA EDITING FACTOR 2 (MORF2) in plant growth and development. Initially recognized as a plastidial RNA-editing factor involved in retrograde signaling (RS), recent interactions of MORF2 with GENOMES UNCOUPLED 1 (GUN1) and the unviable phenotypes of morf2 mutants suggested that MORF2 might have dosage dependent functions or roles beyond RNA editing. Using an inducible Clustered Interspaced Short Palindromic Repeat interference (iCRISPRi) approach, we successfully reduced MORF2 transcripts in a controlled manner. This reduction led to MORF2-dosage dependent RNA-editing errors and stimulated the expression of stress-responsive genes, triggered plastidial retrograde signaling, repressed ethylene signaling and skotomorphogenesis, and increased the accumulation of hydrogen peroxide. These findings, along with previous discoveries, suggest that MORF2 is a crucial regulator involved in plastidial metabolic pathways, and its reduction can activate multiple retrograde signaling molecules, possibly involving reactive oxygen species, to adjust plant growth.

Grapevine (Vitis) is the world’s most valuable fruit crop, therefore, reducing yield losses to stressors is paramount. Vitis labrusca, a wild North American grapevine, is well adapted to its local environment, exhibiting stout pathogen resistance. Meanwhile, Vitis vinifera grapevine, grown worldwide for winemaking, is native to Europe and is highly susceptible to biotic stressors, particularly fungal and insect pests. V. labrusca has been long utilized in Vitis breeding programs to imbue resistance. Therefore, in this dissertation, we determined if V. labrusca acc. ‘GREM4’ was more insect herbivory resistant than V. vinifera cv. ‘PN40024’ and investigated the morphological, genetic, and metabolomic factors which may contribute to resistance. In an herbivory choice assay, Japanese beetles (Popillia japonica), a major pest of grapevine, preferred to feed upon ‘PN40024’ compared to ‘GREM4’. Further, increased leaf area was consumed on ‘PN40024’ compared to ‘GREM4’ in a time course (30min, 1h, and 4h) feeding assay. These results reported ‘GREM4’ is resistant to Japanese beetle herbivory compared to ‘PN40024’. To determine morphological adaptations that may impact defense, trichomes were next investigated. Trichome densities were greater on ‘GREM4’ compared to ‘PN40024’ leaves. In trichome-focused herbivory studies, beetles exhibited a preference for lower trichome density sides of leaves and, when provided tissues with equal trichome densities for both ‘GREM4’ and ‘PN40024’, more leaf tissue was still lost from ‘PN40024’ compared to ‘GREM4’. These results report that trichomes play a role in resistance but are not the sole factor. Therefore, we conducted a comparative transcriptomic analysis to identify differences in gene expression upon insect herbivory between the two species. When comparing constitutive expression differences prior to insect herbivory, genes with greater expression in ‘GREM4’ were enriched in secondary metabolite biosynthesis while enrichment in genes related to plant-pathogen interactions were identified in both species. Upon insect herbivory, the number of significantly differentially expressed genes (DEGs) was lowest in ‘GREM4’ at 30min and highest at 4h while the opposite was observed in ‘PN40024’. By 4h, many defense-related DEGs were identified in ‘GREM4’ compared to relatively few in ‘PN40024’. Systemic responses revealed a greater number of DEGs related to defense and signaling in ‘GREM4’ compared to ‘PN40024’. In both herbivory and systemic responses, flavonoid, phenylpropanoid, acyltransferase, and signaling-pathway genes were identified in greater numbers, or exclusively, in ‘GREM4’ compared to ‘PN40024’. To determine the impact of these transcriptomic alterations on metabolite levels, a comparative untargeted metabolomic study was conducted. Constitutively higher levels of metabolites, such as flavonoids, phenylpropanoids, and terpenes, were identified in ‘GREM4’ compared to ‘PN40024’ leaves while, after 1h of herbivory, a greater number of significantly differentially accumulating metabolites (DAMs) were identified in ‘PN40024’ compared to ‘GREM4’. Constitutively and inducibly increased levels of metabolites with insect repellent and insecticidal properties were observed in ‘GREM4’ compared to ‘PN40024’. Candidate genes and metabolites reported may be employed in future functional studies to determine their impact on resistance. Findings presented herein will inform research endeavors to increase insect herbivory resistance of Vitis, and likely other crops.

Field pennycress (Thlaspi arvense L.) is a winter annual with extreme cold hardiness and seed oil properties desirable for sustainable aviation fuel production. Integration of pennycress as an off-season biofuel cash crop into Midwest corn and soybean rotations could lead to the production of 1 billion liters of seed oil annually, therefore boosting farmer revenue and offsetting carbon emissions. Pennycress fields are vulnerable to heavy spring precipitation events, which can lead to waterlogged soils where the root system is submerged under water. However, it is unknown if growth, development, or yield of pennycress is affected by waterlogging at the reproductive developmental stage which occurs during April. This work aimed to characterize the morphological and transcriptomic responses of pennycress under one week of waterlogging during the reproductive stage. This was done with two core pennycress lines: one is the reference genome (MN106), and one is commonly used for gene editing (SP32-10). Additionally, natural populations of pennycress with predicted variation in soil water availability were included. One week of waterlogging at the reproductive stage under controlled conditions significantly reduced total seed weight in seven accessions, including SP32-10, whereas three accessions did not have reduced seed weight, such as MN106. Therefore, natural phenotypic variation in waterlogging responses existed between the two core lines, so they were further investigated to determine the transcriptomic responses contributing to waterlogging tolerance. Twice as many genes were differentially expressed between waterlogged and control roots in MN106 (3,424 genes) compared to SP32-10 (1,767 genes) after one week of waterlogging at the reproductive stage. Functional enrichment analysis of upregulated differentially expressed genes in both lines revealed Gene Ontology (GO) terms associated with hypoxia and decreased oxygen, including genes involved in alcoholic fermentation and glycolysis. Compared to SP32-10, MN106 waterlogged roots exhibited stronger upregulation of genes involved in hypoxia and glycolysis, as well as strong downregulation of cell wall biogenesis genes. This indicates a better ability of MN106 to respond to the severe energy crisis invoked by waterlogging, possibly by strongly activating anaerobic responses and limiting growth to conserve energy. Lastly, to functionally test the roles of HRE2 and SUS1 in waterlogging tolerance, which were highly expressed in waterlogged roots, EMS mutagenesis lines were waterlogged at the reproductive stage for one week. These two mutant lines were highly sensitive to waterlogging and had significantly reduced seed weight compared to controls, supporting the involvement of these genes in the response and adaptation to low-oxygen stress in pennycress. This research has identified phenotypic and transcriptomic variation in waterlogging responses in pennycress, leading to the identification of candidate genes and pathways involved in waterlogging tolerance. This work provides a foundation for understanding root waterlogging responses in pennycress, which will be useful for breeding climate-resilient pennycress varieties to improve yield following flood events and to expand pennycress production onto marginal lands.

Understanding the physiological basis of variation in seed composition is critical for optimizing soybean seed composition. Also, studying dry matter accumulation and nitrogen uptake, partitioning, and redistribution in soybeans is crucial due to its direct impact on seed yield levels and overall plant productivity. This study aimed to test the primary proxy influencing seed protein and oil content, which is their accumulation rate determined by the assimilate supply per seed. The second objective was to comprehensively understand soybean dry matter (DM) accumulation and nitrogen (N) uptake, partitioning, and removal patterns within the canopy. Field experiments were conducted using two soybean varieties, Hoegemeyer LL2841 (high-protein concentration) and Pioneer P27A17X (low-protein concentration), planted at early, mid, and late dates. The first study showed that at the whole plant level, protein and oil accumulation rates accounted for over 51% of the variation in seed component contents, with leaf area per seed at the R5.5 stage explaining more than 66%. However, within individual canopy strata, the relationship between assimilate supply and accumulation rates was less consistent. Protein accumulation rates significantly influenced protein content in strata 1, 3, and 4, while oil accumulation rates were significant only in strata 4 and 5. The second study indicated that whole plant accumulation of DM and N is minimal until approximately R1, then increases rapidly to the peak near R3 for all treatments. After R5, vegetative organs (leaves and petioles) generally began remobilizing DM and N to seed, whereas stems began DM and N remobilization at R6, possibly supporting greater DM remobilization to the seed. Within strata, peak accumulation of DM and N shifted from R1 to R7 from the bottom of the plant (strata 1) to the top (strata 5). Moreover, vegetative organs varied in their time of maximum DM or N accumulation, and when remobilization of DM or N to seed was initiated. Our results showed that at the whole plant level, seed N accumulation primarily relied on remobilization from vegetative organs rather than on continued uptake. Strata 2 and 3 contributed most of remobilized N to seed. Advisor: John Lindquist

Coastal forested wetlands are identified as some of the highest-priority ecosystems for climate change mitigation. Here, we present two studies conducted in these unique ecosystems. The first assessed changes in aboveground vegetative C stocks along the estuarine-riverine gradients of the Winyah Bay in South Carolina and the Savannah River in Georgia, using a space-for-time substitution to increase understanding of the impacts to upper estuarine forested wetlands from relative sea level rise. Standing C stocks on the Savannah River averaged 183.6 Mg C ha-1, while sites on the Winyah system averaged 162.3 Mg C ha-1. The largest aboveground C pools were found at sites furthest upriver, decreasing downstream as salinity rose. These data, alongside predicted sea-level rise suggest a marked decrease in future aboveground C stocks in forested wetlands situated in and around tidal estuaries. Our second study investigated the radial and azimuthal patterns of sap flow in species (baldcypress and water tupelo) common to the freshwater swamps of the southeastern US, suspecting that these diverse species may exhibit similar sap flow patterns under permanently flooded conditions. Granier-style thermal dissipation probes were used to measure daytime sap flux at 15 mm and 25 mm depths into the active xylem of both species to assess radial patterning and at each cardinal direction on the bole of each species to assess azimuthal patterning. Analysis of sub-daily time series data found significant (p < 0.05) effects on sap flux by time of day, month, species, and depth. We did not find any differences between the two depths with water tupelo (p > 0.5), but average baldcypress flux rates at 15 mm were significantly higher than those at 25 mm (p < 0.0001). Azimuthal patterns were not detected for either species, contradicting previous studies. This study supports the treatment of azimuthal sap flow variation as a random variable rather than one with predictable directionality, and demonstrates that common radial sap flow patterns may not be generalizable across diverse environmental settings. Because our study found temporal variation in sap flux rates, the inclusion of these temporal factors could improve modeling estimates of individual tree water use.

Proteomics, particularly mass spectrometry (MS)-based proteomics, has become an essential tool in understanding biological complexity and function at the molecular level. However, a significant fraction of spectral data generated in these studies, often referred to as the "Dark Matter of Proteomics'', remains unexplored and unidentified, concealing potentially vital biological insights. This dissertation addresses the challenge of uncovering this dark matter through the innovative use of computational techniques to enhance peptide identification and quantification in Kalanchoë fedtschenkoi, a model organism for Crassulacean Acid Metabolism (CAM). The research employs a quantification-centered approach to MS data analysis, leveraging both MS1 and MS2 spectral data to identify and quantify peptides that traditional methods fail to detect. This study elucidates the methodological advancements necessary for decoding complex proteomic data, focusing on high-quality unidentified spectra that could reveal new aspects of plant biology and stress responses. Our findings significantly expand the identified proteome of K. fedtschenkoi, demonstrating that many previously unidentified spectra are indeed derived from peptides with modifications or from unexpected biological sources. The dissertation presents a comprehensive bioinformatics framework that combines existing software tools with novel algorithms to improve the identification rate of MS spectra. Moreover, it highlights the critical role of these unidentified spectra in understanding biological processes, suggesting that they contain modifications and variants crucial for plant adaptation to environmental stresses. This work not only advances our understanding of the proteome of CAM plants but also contributes methodological improvements that can be applied to other proteomics studies, aiming to reduce the volume of unidentified spectral data. The insights gained from this research could lead to new biological discoveries and enhance our understanding of proteomic complexity in other biological systems.

Recent studies show root trait plasticity within species, suggesting differences in root response to ecosystem heterogeneity. The multi-axis root economic spectrum framework (RES) proposes that most root trait variation is explained by phylogeny and ecosystem conditions. We evaluated architecture and morphology in tree species in a long term, nitrogen (N) x phosphorus (P) fertilization study. Root traits differed between taxa and responded to nutrient addition. Red maple roots were less branched, produced fewer tips, and were thicker than roots of the species beech and birch. In both taxa, branching density, total root tips, average diameter, and the root length fraction in the 0-0.25 mm diameter class were lower in response to P. Most of these effects were reversed in response to N+P. Our findings show these species shift root growth to acquisitive traits when P limitations is alleviated but become conservative in response to N+P. This highlights the need to consider whether root growth is single or co-limited in ecosystems due to different effects of N x P interaction on root traits. We must continue refining the multi-axis RES framework to better account for within species plasticity in response to ecosystem conditions.

Abstract: Plants have a remarkable ability to proliferate roots and increase nutrient uptake within nutrient patches in the soil. This behaviour, known as root foraging, describes this ability and what factors may influence or modify this response. It has been observed that plants integrate multiple environmental cues to inform their root foraging behaviour, resulting in non-additive responses to novel combinations of stimuli such as nutrient distribution and the presence and identity of neighbouring plants. These responses are highly species-specific and context- dependent. While the effect of nutrient distribution and competitors on foraging behaviour has been studied quite comprehensively in both plants and animals, the impact of injury on the foraging behaviour of plants remains largely unexplored. In plants, defoliation can be considered a form of injury. Although both plants and animals need to forage for nutrients, it is unclear if they respond to injury in a similar manner. The many physiological and ecological differences between plants and animals, as well as their distinct interactions with the environment, cannot be disregarded. Therefore, it is essential to experimentally test the assumption that they may behave in the same way. To experimentally evaluate the impact of injury and its severity on root foraging behaviour over time, we conducted a study using the model organism, common sunflower (Helianthus annuus L.). Individual sunflower seeds were planted in experimental arenas that allowed us to observe root growth over time. These arenas contained soil with low nutrient levels and a nutrient-rich patch, enabling us to measure root foraging responses to heterogenous nutrient distribution in the soil. Defoliation treatments were applied at two different severities: half clipped and fully clipped, alongside a control treatment in which no defoliation occurred. Defoliation was found to suppress the overall root length grown, but had no effect on root foraging precision. Over time, root foraging precision decreased from an initially high proliferation response to nutrients to an agnostic response to nutrient patch by the fourth week of growth. Other measures of root morphology, namely average root diameter and average root length, exhibited no changes over time or in response to defoliation. The average root diameter was consistently larger within the nutrient patch, while average root length remained equivalent in both the patch and background soil throughout the study. These findings generally align with assumptions based on optimal foraging and optimal defence theory. However, they also highlight the influence the ecological and physiological differences between plants and animals can have on the applications of optimality theories. Additionally, this study emphasizes the importance of considering the various different measures of root foraging behaviour and the timing of data collection. Different measures and their timing yielded distinct results, and only when all measures were compared could a comprehensive understanding be obtained. Ultimately, comparing the possible effects of timing and types of measures on a study’s findings and experimentally testing the applicability of established theories to novel organisms enhances our understanding of how past and future studies can be effectively compared. This includes considering the chosen measures and timing of data collection, as well as the theories themselves and their applicability in future research.

Transcription factors involved in regulating plant cell wall biosynthesis are diverse and come from various families, including but not limited to AP2/ERF, MYB, NAC, bZIP, and WRKY. These TFs play crucial roles in modulating the expression of genes responsible for cell wall synthesis, composition, and structure, thereby influencing plant development and responses to environmental stimuli. Using gene association networks (GAN) analysis, four transcription factors co-expressed with cellulose synthases and glycosyltransferases for xylan biosynthesis were identified, namely, Os06g0194000 (OsERF071), Os02g0782700 (OsERF070), Os09g0434500 (OsERF072) from the AP2-ERF family, and Os08g0549000 (OsMYB102) from the MYB family. Loss-of-function allelic mutants were created using CRISPR/Cas9 technology. Allelic mutants that are homozygous and Cas9-free were evaluated morphologically, physiologically, and biochemically in the T2 generation. Mutants in Os06g0194000 (OsERF071) exhibited the most significant alterations compared to the CRISPR control plants, prompting the generation of overexpression lines for this gene to enable a more comprehensive functional analysis of this gene. Mutants lacking OSERF071 were taller plants with larger seed sizes, panicle lengths, thousand grain weights, biological yields, and grain yields compared to the CRISPR control plants, while the opposite phenotypes were observed in overexpression lines. Additionally, the allelic mutants exhibited higher content of lignin in their tissues compared to the control and showed higher expression levels of the OsCesA genes related to secondary cell wall biosynthesis compared to those linked to primary cell wall biosynthesis, suggesting OsERF071 may function as a repressor of lignin biosynthesis during primary cell wall deposition. Taken together, my results suggest that OsERF071 may function as a repressor of secondary cell wall formation in tissues that are dividing and growing. Another gene of interest was Os09g0434500 (OsERF072). Allelic mutants in OSERF072 exhibited reduced grain yields and early flowering phenotypes. Notably, these mutants displayed decreased lignin content compared to the control plants. In contrast, allelic mutants in the Os02g0782700 (OsERF070) and Os08g0549000 (OsMYB120) genes were comparable to control plants. While this study sheds light on various aspects of the role of these transcription factors in regulating cell wall biosynthesis, further experiments are needed to identify target genes of these transcription factors.

Heteroxylans (HXs) are major hemicellulosic polymers, that provide structural integrity of plant cell walls which directly impact the growth and development of the plant. The backbone of HXs is comprised of β-linked xylose (Xyl) residues with different substitution patterns (acetyl, glucuronic acid/methylglucuronic acid [GlcA/[Me]GlcA] and/or arabinofuranosyl [Ara] side chains). In Arabidopsis, glucuronidation of HXs is mediated by GlucUronic acid substitution of Xylan (GUX) enzymes which are members of the GT8 family (CAZy). Reduced GlcA/[Me]GlcA side chains in HXs have been found to improve saccharification by reducing biomass recalcitrance and improving biofuel production. Therefore, identifying GUXs is of importance, offering insights into enhancing biofuel production and ensuring agricultural sustainability. Recently, GUXs have been identified in other species like conifers, Brachypodium and sugarcane. However, several outstanding questions about HX glucuronidation in grasses remain unanswered. Thus, this work focuses on the understanding of HX glucuronidation in rice, a significant cereal crop renowned for its nutritional value and substantial biomass, holding great potential for biofuel conversion and food security challenges. Through comprehensive phylogenetic and bioinformatics analyses, I identified three OsGUXs in rice, designated as OsGUX-A, OsGUX-B, and OsGUX-C. These OsGUX proteins exhibited structural conservation with counterparts from various species and harbored specific motifs and domains characteristic of GUX enzymes. Subcellular localization studies confirmed their presence within the Golgi apparatus, consistent with the site of HX biosynthesis. Using in vitro assays, I demonstrated that these OsGUXs have glucuronosyltransferase activity and exclusively utilize UDP-GlcA as a substrate. Further investigation through genetic complementation of Arabidopsis gux1/2/3 mutants, characterized by HX lacking GlcA/[Me]GlcA side chains, indicated that OsGUXs possess the capability to add GlcA to HXs, albeit to a limited extent. However, this complementation did not reach up to 100% of the wild-type level, suggesting differences in glucuronidation mechanisms between monocots and dicots.To elucidate the physiological function of OsGUXs, several CRISPR/Cas9-mediated allelic knockout mutants were generated in rice. Phenotypic analysis of these mutants indicated minor alterations in plant morphology and growth. 1H NMR analysis of HX structure from these mutants revealed a notable reduction in GlcA content in osgux-a/b/c triple and osgux-c single knockout mutants, while an increase in GlcA content was observed in osgux-a and osgux-b single knockout lines. Based on these findings, I conclude that OsGUXs do not compensate for each other. OsGUX-A and OsGUX-B may contribute to the formation of GlcA-rich domains on the HX backbone that are resistant to endoxylanase, whereas OsGUX-C may be involved in generating well-spaced GlcA domains that are susceptible to endoxylanase. Not only do my findings provide insights into the mechanisms of HX glucuronidation in rice, which advances our understanding of plant cell wall biosynthesis, but they also hold promise for optimizing biofuel production strategies.

Protein degradation through the Ubiquitin (Ub)-26S Proteasome System (UPS) is a majorgene expression regulatory pathway in plants. In this pathway, the 76-amino acid Ubproteins are covalently linked onto a large array of UPS substrates with the help of threeenzymes (E1 activating, E2 conjugating and E3 ligating enzymes) and direct them forturnover in the 26S proteasome complex or through autophagy-mediated degradation.The S-Phase Kinase-Associated Protein 1 (Skp1), cullin (CUL)1, and F-box (FBX)protein (SCF) complexes have been identified as the largest E3 ligase group in plants.Since the FBX proteins recognize and determine the specificity of SCF substrates, mucheffort has made to characterize their genomic, physiological, and biochemical roles in thepast two decades of functional genomic studies. The co-immunoprecipitation failed tocapture transient interaction while the yeast two-hybrid only test protein interaction inyeast cell instead of plant tissue. Therefore, the sheer number and high sequence diversity ofthe FBX gene family demands new approaches to uncover unknown functions. In thisresearch, first, from the proteomic level, I developed and tested a proximity labeling andco-immunoprecipitation approach for determining the proteome of active FBX proteinsand their associated complexes in Arabidopsis. We demonstrate that ASK1-TurboID isnot fully functioning, which led us to discover a novel antagonism between biotinylationand ubiquitylation in regulating protein stability in vivo. This discovery lowers theeffectiveness of proximity labeling in ubiquitylation studies. Second, from the FBX level,I characterized a new FBX protein that is involved in reproductive development. Themutant was created using CRISPR/Cas9. And it was confirmed that mutation of this FBXgene impacted seed development. The potential substrates were identified and confirmedusing yeast two-hybrid and split-luciferase assay. Third, from the substrate level, Ilabeled phytochrome A (phyA), a master red and far-red photoreceptor in plants, with agenetically engineered biotin ligase, TurboID, in vivo for searching putative unknownFBX proteins that may target phyA for degradation. I succeeded to apply phyA-TurboIDfusion for complementing the far-red response of phyA-211 null mutant allele, suggestingan efficacy of PL in characterizing single ubiquitylation pathways. This study provided anew evidence about the limitation of PL in ubiquitylation studies, discovered a newantagonistic pathway of biotinylation, and developed effective genetic materials andtheoretical guidance for future proximity labeling based characterization of ubiquitylationpathways.

Functional studies of the ubiquitin (Ub)-26S proteasome system (UPS) have demonstrated that virtually all aspects of the plant’s life involve UPS-mediated turnover of abnormal or short-lived proteins. However, developmental characterization of the UPS, including in seeds and fruits, remains scarce. Unfortunately, early termination of embryogenesis limits the scope for characterizing the UPS activities in reproductive organs. In this dissertation, I utilized a biochemical approach to tackle the developmental role of UPS in plants, using Arabidopsis thaliana (Arabidopsis hereafter) as a model. The overarching goal of my research is to unravel the molecular mechanisms of UPS underpinning seed development so that new molecular breeding technologies could be developed to promote seed production. First, I systematically compared expression changes of multiple 26S proteasome subunits along with the dynamics of proteasome activity and total protein ubiquitylation in seedlings and developing siliques of Arabidopsis. Because autophagy plays the second largest role in maintaining proteome stability, I parallelly studied three late-limiting enzymes that are involved in autophagy influx. My experiments unexpectedly discovered that, in opposite to the activities in seedlings, both protein and transcript levels of six selected 26S proteasome subunits gradually decline in immature siliques toward maturation while the autophagy influx rises, albeit in a nutrient-rich condition. I also discovered a reciprocal turnover pathway between the proteasome and autophagy. While the autophagy influx is suppressed in seedlings by UPS-mediated degradation of its three key enzymes, transcriptional reprogramming dampens this process in siliques that in turn stimulates a bulk autophagy degradation of proteasomes. Collectively, my discovery about the developmental changes of the UPS and autophagy activities suggests that they relay the proteome homeostasis regulation in early seed development, which highlights their developmental interactions.In the second objective, I studied the impact of protein ubiquitylation on seed development by overexpressing a polyUb (UBQ) gene. To demonstrate the dosage-dependent impact of protein ubiquitylation, two different UBQ transgenic plants transformed with either hexa(6His-UBQ) driven by Cauliflower mosaic virus (CaMV) 35S promoter or hexa(6His-Tev-UBQ) driven by the endogenous promoter of UBQ10 were studied. Given the nature of different transgenes, I termed the two transgenic plants as HU (for 6His-UBQ) and HTU (for 6His-Tev-UBQ), respectively. I discovered that HU and HTU had contrasting effects on seed yield, as the former exhibited reduced seed size and yield, while the latter showed an opposite phenotype attributed to enhanced silique number per plant. Differences in the integrity and composition of the 26S proteasome assemblies in the two transgenic plants were also notable in comparison with the wild type (WT) plant suggestive of a contributory role of UBQ expression in proteasome assembly. Since large protein complexes or aggregates and damaged cellular organelles are targeted by protein ubiquitylation for autophagy-mediated degradation for nutrient recycle, a compromised carbon starvation response was found in both HU and HTU seedlings, further suggesting the importance of UPS in regulating plant development, including seed development. In the third objective, I aimed to apply an affinity purification-based mass spectrometry (MS) analysis to identify active Skp1-cullin (CUL)1-F-box (SCF) Ub E3 ligase complexes and substrates that may differentially regulate plant vegetative and reproductive growth. I have identified 24 F-box proteins and 590 potentially SCF substrates, providing an exciting direction to further explore the role of SCF-meditated protein ubiquitylation in plant development and growth.

Caryophyllaceae flowers have a wide range of morphological appearances, particularly in regards to their stamens and petaloids. In order to determine how these structures evolved, I reviewed the phylogenetic relationships, mature morphology, and developmental pathways of flowers in the family. I concluded that in addition to an antesepalous whorl of stamens, there was likely a whorl of some ancestral structure in the alternisepalous position. This structure may have been a petal, a stamen, or a transitionary staminode, but it diversified in various lineages into the numerous forms we see today.

A plant’s survival is largely impacted by its ability to sense and respond to gravitational changes, such as the changes that occur in spaceflight. However, the gravitropic signaling pathway is not fully understood. In the BRIC-20 experiment aboard the International Space Station, the Arabidopsis proton pump AHA2 was found to be differentially phosphorylated in microgravity compared to ground controls. AHA2 is hypothesized to be involved between plant hormone movement and differential growth during gravity signaling. When subject to reorientation, aha2 mutant seedlings had increased root curvature compared to wild type. Furthermore, wild type seedlings treated with fusicoccin, a phytotoxin that increases AHA2 phosphorylation and activity, had increased root curvature compared to untreated plants. To determine the role of AHA2 phosphorylation in gravitropic signaling, several lines of transgenic Arabidopsis with modifications in phosphorylation sites are being developed. The transformants will then be phenotyped for altered gravity response, showing if the phosphorylation of the AHA2 protein is involved in a plant’s gravity response. These findings will further knowledge on the molecular mechanisms of gravitropism to develop space-tolerant plants for life support in spaceflight and for habitation of the moon, Mars, and beyond.

As land area in grain crop production increases in the US Midwest, perennial pasture availability decreases but the demand increases. Matching the seasonal growth patterns of C3 and C4 plant species to grow forages that are complementary allows producers to fill production gaps in otherwise monoculture systems and possibly increase overall productivity. One way to fill the forage production gap of C3 grass dominated pastures is by producing a warm-season, annual forage in a separate field. However, with continual reductions in land availability, this may not be an option. Using two studies, our objectives were to improve forage crop production within existing perennial pastures and in historical cropland converted to annual pastures. We also wanted to compare the effects of N fertilized grass monocultures to non-N fertilized grasses mixed with legumes to see if N fertilization could be replaced by legumes. To improve the total forage production within smooth bromegrass pastures, we sod-seeded sorghum × sudangrass but found too large a reduction in smooth bromegrass forage mass the following year with no improvement in forage production. To sustainably improve forage production and distribution within land area under annual forage production, we tested the viability of a double cropped forage system using fall planted triticale cut once in the spring followed by pearl millet cut once in the summer and compared it to a perennial system containing smooth bromegrass. We found that timely precipitation was crucial to the success of the annual system and when abundant, the double cropped annual forage system produced most of the forage during the summer. This contrasts with smooth bromegrass pastures, which produced the greatest during the spring. Nitrogen fertilization and mixed grass-legume produced similar forage mass in smooth bromegrass pastures, but mixed grass-legume pastures produced less mass in the annual forage system.

Salt pollution and soil salinization via secondary pathways is a global problem. One practice which contributes to increased salinity in soils and other natural systems is the application of road deicers in colder regions. Increased salinity in soils negatively affects plant growth and microbial activity. In an effort to decrease the amount of salt applied to roads, salts are applied as brine. Liquid organic additives are incorporated into these brines to foster better adherence of the salt to impermeable surfaces. One common additive is beet juice, a byproduct of sugar extraction. Beet juice contains high levels of phosphorus and organic sugars, and thus might have beneficial effects on soil microbial activity and plant growth, mitigating some negative effects of salt. To investigate the combined effects of NaCl and beet juice on germination and early growth of plants a series of treatments comprising combinations of no, ‘low’(2g), or ‘high’ (7g) NaCl and no, ‘low’(5mL), or ‘high’ (20mL) beet juice were administered. To investigate treatment effects on microbial activity, the same treatments were applied to equivalent non-plant treatments. High salt additions reduced seed germination success, delayed day of germination, decreased leaf count, and decreased final height among R. sativus. Additions of beet juice had no observable positive effect on plant growth, and might have exacerbated negative effects of salt stress. High and low beet juice additions increase soil respiration, indicating that the microbial community responded to beet juice additions, but without improving seed germination success or growth. The results from this study highlight the importance of studying co-contaminants that affect ecological communities.

Embryophytes have undergone numerous re-invasions to aquatic habitats, impacting their morphological evolution. Despite the prominence of these transitions, their phylogenetic implications and morphological adaptations are just beginning to be investigated, especially as molecular methodologies become more accessible. We investigate these phenomena using the genus Fissidens, a diverse group of mosses encompassing both terrestrial and aquatic species. Our study integrates phylogenetic methods with morphological analyses to elucidate the evolutionary patterns within Fissidens and explore correlations between habitat transitions and morphological traits. Through extensive taxon sampling from herbarium specimens, DNA extraction, and sequencing, we construct robust phylogenetic trees using concatenated and coalescence methods. Our results reveal the non-monophyly of historically defined subgroups within the genus Fissidens, challenging prior classifications. Furthermore, ancestral state reconstructions suggest there have been multiple independent transitions to aquatic environments within the genus. Morphological analyses indicate that although variations in leaf length correlated with habitat moisture, phylogenetic relatedness diminishes this correlation. These findings better our understanding of the complexity of evolutionary transitions in embryophytes and emphasizes the need for further studies to refine taxonomic classifications and better understand morphological changes in response to ecological shifts. This study contributes to wider discussions on the evolutionary dynamics of aquatic transitions in both bryophytes and, more broadly, embryophytes and their ecological significance.

The dispersal of pollen from genetically engineered (GE) crops can lead to unintended transgene flow, especially in wind-pollinated and obligate-outcrossing species such as switchgrass (Panicum virgatum L.). Various bioconfinement approaches have been studied for their potential to eliminate transgene flow from GE crops including selective male sterility, which aims to inhibit the germination of transgenic pollen grains. In this study, pollen-specific promoters controlling the expression of the EcoRI endonuclease, interrupted by a catalase intron, was evaluated for its efficacy to produce sterile pollen. The TaPSG719, PvPS1, Osg6B, OsRTS, and Zm13 promoters were assessed for pollen-specific expression patterns, none of which have previously been characterized in switchgrass. Here, I report relatively high EcoRI transcript abundance in pollen/anther tissue compared to the switchgrass Ubq1 reference gene when EcoRI is driven by the Zm13 (10% ± 0.9%) and PvPS1 (6% ± 1.3%) promoters. However, the TaPSG719, Osg6B, and OsRTS promoters did not significantly differ from nontransgenic switchgrass in terms of EcoRI transcript abundance in pollen/anther tissue. Furthermore, the highest-expressing transgenic line of the Zm13:EcoRI and PvPS1:EcoRI constructs were used for pollen germination experiments. These lines were presumed to contain a mixture of transgenic and nontransgenic pollen and were observed to exhibit similar pollen germination percentages compared to nontransgenic switchgrass pollen. Upon further investigation, it was determined that the catalase intron was incorrectly spliced from the mRNA, likely resulting in a nonfunctional protein when translated, which may explain why similar pollen germination rates were observed between the transgenic lines and control. In addition to these findings, I observed statistically significant phenotypic differences in many transgenic switchgrass lines when compared to nontransgenic control switchgrass. The most commonly observed adverse trait is decreased plant height, followed by decreased leaf blade width, decreased tiller diameter, and increased tiller count. However, off-target EcoRI expression did not appear correlative with plant height (r= -0.08, p= 0.4134) or leaf blade width (r= -0.23, p= 0.0133), suggesting alternative explanations such as inapt T-DNA integration sites and the high genetic diversity of switchgrass. Overall, the effectiveness of pollen-targeted EcoRI expression to produce sterile pollen was not determined. However, this study identified the Zm13 and PvPS1 promoters as strong candidates for male-specific gene expression and provided valuable insights for the design and production of genetically engineered switchgrass.

Globally, grasslands are declining in range, which is leading to a decline in grassland-dependent taxa. These declines can be attributed to a variety of issues, a major one being the encroachment of woody species into historic grasslands. There is a new framework for strategizing grassland management, referred to as “Defend the Core,” which calls for proactive management of intact grasslands, meaning conserving remaining grasslands by preventing woody encroachment in the first place. To successfully defend the core, it is imperative that we develop rapid methods to identify intact grassland cores for management priority. One method that can be used for identifying grassland cores is identifying and tracking spatial regimes, which can be done using the relatively new metric of spatial covariance. Spatial covariance can measure the degree of coexistence between two vegetative functional groups, essentially quantifying boundaries between two cover types. To identify grassland regimes, or intact grassland cores, we can calculate the spatial covariance between tree cover and grass cover. However, this metric was recently developed, and knowledge gaps remain. This metric has never been tested in grasslands of the southeastern United States and has not been used as a covariate for occupancy probability modeling. Additionally, spatial covariance has never been related to established on-the-ground metrics of vegetation structure. For this thesis, we used the Rangeland Analysis Platform cover data to calculate spatial covariance between tree and grass cover in historic grassland sites across the Mississippi Alluvial Valley and the Arkansas River Valley in central and east Arkansas. These sites have undergone varying management interventions and are highly fragmented in comparison to rangelands of the western US, where spatial covariance has previously been tested. In Chapter I, we related spatial covariance to a variety of established metrics for quantifying vegetation structure. We collected vegetation data at our study sites and used generalized linear models to understand the relationship between each vegetation measurement and spatial covariance. We found that there were no strong relationships between on-the-ground vegetation metrics and spatial covariance, only further emphasizing that spatial covariance is identifying and measuring something that is not captured by many traditional field-collected vegetation metrics. In Chapter II, we used spatial covariance in an occupancy framework to understand avian responses to spatial covariance and boundaries of grassland cores. We used a multi-species occupancy model to predict occupancy probability of the grassland bird community and individual grassland bird species and mapped these results across a known grassland core. We found that all grassland birds included in our models avoided areas with negative spatial covariance, or boundaries between grass and tree cover, and were more likely to occupy intact grassland cores that are free of boundaries. In this thesis, we delve deeper into the recently developed spatial covariance metric. We find that this metric is promising for identifying grassland cores from an avian perspective, which only helps better strategize management and protection of grasslands.

Beginning in 2012, the local non-profit, The Lincoln Botanical Garden, began organizing the conceptual design and creation of a glass conservatory within a botanical garden in Lincoln, Nebraska. These proposed urban enhancements seek to fill a gap in garden amenities in the city by providing year-round access to public horticulture programming and education. This project, Public Horticulture: Process and Design of the Lincoln Botanical Garden, documents these efforts and makes design recommendations for a themed Glacial Erratic Garden that could be constructed within the botanical garden. Advisors: Ellen Paparozzi and Sam Wortman

Roadsides provide an abundant opportunity to increase connectivity of fragmented landscapes with diminishing floral resources for pollinating insects. The ecological value of these sites is often overlooked as quality habitat for pollinators, particularly monarch butterflies, which have been experiencing severe declines due to loss of habitat and loss of milkweeds that provide food for their larvae. Land managers across the nation are realizing the potential of roadsides to provide high quality floral resources for the benefit of insects and other wildlife. Current wildflower seed mixes used by state transportation departments are often low diversity and may only be implemented following road construction with no follow-up management. Mowing is often used on roadsides to manage vegetation for safety and aesthetic reasons. The timing and frequency of mowing can impact the density and diversity of wildflowers present on a site. This research seeks to determine the efficacy of seeding a diverse, native wildflower seed mix into backslopes with well-established perennial grasses. We monitored two sites in southeast Nebraska in 2021 and 2022. Two mowing regimes: October pre-seeding mowing and July post-seeding mowing were studied to determine the effects that mowing may have on existing perennial grasses. Our results indicate that seeding wildflowers is effective at increasing wildflower density, diversity, and floristic quality of a site. Milkweed abundance also increased because of seeding. Mowing dormant vegetation in October before seeding appears to increase forb cover and floristic quality of one site, particularly in the first season after planting. This benefit of pre-seeding mowing appears to stem from a reduction in vegetative litter in the plots. Post-seeding mowing provided no benefit to wildflower density or diversity. Neither mowing treatment led to a reduction of perennial grass cover by the second season of the study, confirming that grasses can recover quickly following disturbance. The temporary reduction of litter following October mowing, however, may enable forbs to germinate in the spring following seeding. Advisor: John Guretzky

Nutrient limitation drives forest ecosystem processes by altering net primary production, belowground (C) investments, and microbial activity. As such, the availability of essential macronutrients, such as nitrogen (N) and phosphorus (P), can severely impact plant growth, microbial decomposition, and the strength of plant-microbial interactions. However, our understanding of plant and microbe responses to changing nutrient availability, and the extent to which nutrient availability affects plant-microbe interactions, remains somewhat limited. Therefore, in this dissertation, I aimed to investigate nutrient limitation on a plant, microbe, and plant-microbe interaction level. First, to connect the phenotypic responses of nutrient-limited plants to key molecular mechanisms, I used a multi-omics approach to examine the responses of black cottonwood root and shoot transcriptomes, proteomes, and metabolomes to P deficiency. Next, to examine the susceptibility of potentially vulnerable soil C stocks to regional and global changes, I investigated the effects of removing microbial limitations on soil organic matter (SOM) decomposition in temperate forest soils after long-term N fertilization and subsequent soil C gains. Finally, to understand the responses of plant-microbial interactions to changes in nutrient availability, I compared forest stands dominated by arbuscular mycorrhizal (AM) species to forest stands dominated by ectomycorrhizal (ECM) species in their recovery from long-term N fertilization. In my first chapter, I showed that different cottonwood -omes may show either coordinated or conflicting responses to P limitation, highlighting the importance of multi-omics integration in plant nutrient stress studies. Next, I found that microbial activity remained suppressed in soils after long-term N fertilization even when N-induced limitations on decomposition, such as soil pH, plant C investments, and soil warming, were removed. Finally, I found that AM dominated stands may recover more rapidly than ECM stands after excess N inputs ceased. Given the declining rates of N-deposition and the increasing rates of nutrient limitation of both plants and soil microbes, my dissertation work linking nutrient limitation with plant and microbial traits has important implications for predicting the future of soil C stocks in Eastern temperate forests of the US.

A transition away from fossil fuel use is necessary for the future of energy production, and biofuels are a renewable energy option that can mitigate the impacts of fossil fuels through lowered carbon emissions and possible carbon sequestration. In order to successfully compete with fossil fuels, the production of biofuels must be sustainable and cost effective. Populus species are a biofuel crop used for bioenergy conversion from lignocellulosic biomass that have been proposed for use in feedstock plantations on marginal lands, which would avoid competition with agricultural production. Two challenges of feedstock production being faced are recalcitrance to chemical processing, and the effects of increasing stress on plant productivity. To address these challenges, genetic manipulation is being used to improve agronomic performance and achieve desired growth and wood property phenotypes. It is important to understand how these modified feedstocks will perform in plantations to ensure they are achieving desired phenotypes and improving production for biofuel use. To understand how transgenic Populus feedstocks perform under conditions like proposed plantations I examined two transgenic field trials addressing the challenges mentioned previously. The first study examined Populus deltoides transformed with genes that showed reduced recalcitrance phenotypes in preliminary greenhouse studies. It was observed that the phenotypes exhibited in greenhouse studies were not present under field conditions, and none of the transgenic lines showed improved growth compared to the controls. One transgenic line overexpressing O-acetyltransferase had an increased S/G ratio, a phenotype indicative of reduced recalcitrance, but had decreased growth compared to controls. The loss of phenotypes could possibly be a result of transgene silencing under field conditions or low target gene expression, but RNA analysis is needed to confirm. The second study of this thesis examined the performance of Populus tremula x alba transformed with heat, salt, and water deficit stress response genes to improve stress tolerance. These genes were paired with a constitutive gene promoter and a stress inducible promoter to examine how the inducibility of target gene expression impacts field performance. This study found that there is no clear difference in performance in the field based on promoter type, and the only significant difference in performance compared to controls was observed in Heat shock factor 2 overexpression lines that exhibited impaired growth in the field. Collectively my research shows that the performance of transgenic feedstocks can differ greatly under field conditions compared to greenhouse performance, and that field screenings are crucial in developing transgenic feedstocks for bioenergy use.

Unprecedented weather events associated with climate change, coupled with extensive anthropogenic stresses, have reduced the effectiveness of traditional forest pest management approaches, which can no longer keep pace with invasive species overwhelming naïve landscapes and native pests experiencing eruptive and expansive outbreaks. Double-stranded RNA-(dsRNA) mediated gene silencing, a type of RNA interference (RNAi), has been coopted for use as a biopesticide against a multitude of agricultural and horticultural pests and could serve as a powerful tool for woody plant protection. While effective initiation of gene silencing and subsequent mortality have been demonstrated in multiple tree pests, effective and efficient methods of delivery and plant compatibility must first be understood. To better understand how dsRNA applied as a root soak is taken up by woody plants, I investigated the translocation and persistence of different dsRNAs in both coniferous and deciduous seedlings. For Chapters 2 and 3, I submerged the roots of deciduous white oak (Quercus alba, L.) and coniferous loblolly pine (Pinus taeda, L.) in a water and dsRNA solution and used end-point RT-PCR to detect the presence of intact, exogenous dsRNA in various tissue types over the course of 7 days. While both seedling types showed rapid uptake and whole-plant dissemination, dsRNA recovery quickly waned over time in loblolly pine while detection decreased only slightly in white oak. This work shows that naked dsRNA can rapidly disseminate throughout woody plants and sheds light on possible differences between coniferous and deciduous trees. Since plants possess intrinsic RNAi machinery that can respond to and defend against invading dsRNA, I annotated RNAi-related proteins (RRP) in loblolly pine and investigated the expression response of their corresponding mRNAs in the presence of dsRNA for Chapter 4. Annotation of RRP in loblolly pine, as well as selected proteins in five Pinus spp., established new sequences for these proteins and highlight the diversity and possible redundancy in some of these vital pathways. Differential expression analysis identified only two isoforms of RRP that were upregulated in dsRNA treated seedlings, demonstrating an overall lack of RNAi response detected. Despite this, pathway enrichment revealed upregulation of autophagy and plant hormone signal transduction pathways, which may be responding to the dsRNA challenge. Knowing how, and to what degree, plants may recognize and respond to insect-specific dsRNA will help to increase compatibility with plant uptake and may reduce plant processing of these insect biopesticides. Finally, in Chapter 5, I began the work to establish transgenic Arabidopsis thaliana (L.) Heynh. for the production of insect specific dsRNA. Despite the successful production of entry clones, I documented unique and erroneous recombination during the LR reaction when attempting to generate the expression clone. This issue with pHELLSGATE12 recombination is previously undocumented but now replicated with multiple entry clones. However, restriction digest procedures may circumvent this issue while also allowing for flexibility in future intron containing constructs. Overcoming the barriers associated with dsRNA deployment, specifically that of practical delivery and efficient production, will help move this technology closer to commercial use. Overall, my data demonstrate the applicability of root delivered dsRNA in both deciduous and coniferous seedlings and its compatibility within woody plant tissue.

Seed-transmissible epichloid fungal endophytes are best known for their roles as defensive mutualists in cool-season grasses. Historically, the discovery of fungal endophytes was driven by investigations of plant toxicity to livestock, followed by extensive study of their alkaloids and protection against insects and nematodes. Epichloae can produce four classes of alkaloids: ergot alkaloids, lolines (saturated aminopyrrolizidines), indole–diterpenes, and peramine. It is increasingly evident that these hereditary symbionts have much more diverse chemical profiles both in individual populations and between them. To this end, differences in chemotypic profiles of these symbionts may translate to different evolutionary and environmental advantages across plant species, as well as influences on phenotypic measurements. In an ecological sense, the chemotypic diversity within the species may reflect frequency-dependent selection for the alkaloids, which can be metabolically expensive to produce. To date, there has yet to be extensive study on the alkaloid profiles of the fungal symbiont Epichloë brachyelytri in the cool-grass host Brachyelytrum erectum. This project aims to initiate the required genomic population analyses to comprehend endophyte frequency and chemotypic diversity among and between populations throughout the state of Kentucky. In a previous survey of B. erectum in the Kentucky Palisades, 50% of the plants had fungal endophytes, with two endophyte genotypes that differed in alkaloid profiles. In the investigation, 21 populations of B. erectum were sampled and the genetic and chemotypic diversity analyzed by high-throughput tiller extraction and multiplex PCR. Biosynthesis genes for ergot alkaloids (e.g., chanoclavine and ergovaline), aminopyrrolizidines (e.g., lolines), and pyrrolopyrazines (e.g., peramine), as well as genes for mating type and phylogenetic barcodes, were interrogated to determine endophyte presence and diversity. Morphological measures for all samples were also assessed, and six populations were sampled for transcriptome analysis. The results of these studies will expand the understanding of how endophyte frequency distributions and genetic diversity can influence functional and phylogenetic diversity.

Due to the ongoing rise in atmospheric carbon dioxide (CO2) concentrations and other downstream effects of climate change, global crop agricultural yields are projected to be severely impacted. To combat this issue, a clear understanding of the molecular mechanisms by which plants are able to respond to increasing CO2 levels is imperative. This study serves to characterize and investigate the roles of several proteins suspected to function in CO2-induced signal transduction in Arabidopsis thaliana and Solanum lycopersicum guard cells. This research was conducted through the construction and screening of multiple mutant plant lines as well as analysis of their stomatal gas exchange responses to imposed shifts in [CO2]. Previous research has revealed the importance of the MAP kinase MPK12 in its role as an inhibitor of the Raf-like kinase HT1, a principal negative regulator of high CO2-induced stomatal closure. However, the mechanism by which this inhibitory interaction occurs is not yet confirmed. Results obtained from this study support previous findings suggesting that the phosphorylation activity of MPK12 is not required for its function in CO2 signaling through gas exchange analysis of a kinase inactive isoform. Additionally, another protein named AtMRK1 is suspected to function in guard cell CO2 signaling in tandem with two previously identified MAPKKK proteins of the same clade, named CBC1 and CBC2. Thus, a cbc1/cbc2/atmrk1 allelic triple mutant for all three genes was developed and screened for in A. thaliana to potentially observe whether AtMRK1 partially complements the activity of CBC1 and CBC2 in future analyses. Lastly, preliminary gas exchange experiments were pursued on mutant alleles of the SlROP9 protein in S. lycopersicum. The preliminary data obtained here suggest that rop9 mutants experience constitutively reduced levels of stomatal conductance with no impairment to CO2 signal transduction, and thus are likely to exhibit increased water use efficiency when compared to the wildtype allele.

Following centuries of exploitation and fire suppression, longleaf pine systems are now the focus of many conservation efforts. Efforts to restore populations of Schwalbea americana L. in longleaf pine savannas have been met with frustratingly low recruitment. While past studies have briefly quantified germination rates for Schwalbea, there have not been any studies yet that truly investigate this plant’s germination requirements. Additionally, there has been little research into characterizing the parasitic relationship between Schwalbea and its various host species. We conducted a germination study in a growth chamber that investigated Schwalbea’s germination rate and time to germinates as response variables using stratification and time between dispersal and sowing as independent variables. We found that a cold stratification significantly influenced germination rates, and after allowing one or two months to pass between dispersal and sowing the seeds, we observed a stronger germination response following stratification, and shorter time to germinate. Additionally, we explored the post-germination development of Schwalbea seedlings at varying levels of host and resource availability. Host presence resulted in greater resource allocation to storage and regenerative organs (i.e. thickened roots and dormant buds) with higher moisture levels amplifying this effect. Knowledge of these germination and seedling development trends may help facilitate future conservation and reintroduction efforts and may be used to inform future studies with the goal of facilitating in situ recruitment.

This thesis delves into the impact of stress on plants amid climate change, exploring strategies for mitigation. The initial focus is on duckweed stress tolerance, evaluating how different ecotypes respond to various stressors over three weeks. The L. minor ecotype from the subarctic climate, Norway, displayed the highest levels of tolerance and plasticity by having significantly larger growth areas compared to all other ecotypes across treatment groups after three weeks. Many samples underwent increases in growth after three weeks of stress exposure compared to just one week of stress exposure. Transcriptomic analysis showed that salt exposure caused repression of genes related to photosynthesis and plastid localization and organization in the first week, along with induction of stress response genes. The processes that were repressed in the first week were induced by the third week, causing growth areas to increase significantly. Copper sulfate exposure induced genes related to ion and metal transportation, as well as homeostasis, which contributed to the significant increase in growth seen in week three in these samples. This study offers that the 7-day ecotoxicity study that is currently used for Lemna minor may not cover the entire stress response. Additionally, as duckweed is used worldwide for many applications such as feed production or phytoremediation, the ecotype utilized may determine the success of the system. Potato plants are also utilized globally, making stress mitigation strategies crucial as climates change. The following chapter discusses the increase of potato stress tolerance by means of Escherichia coli OtsB [trehaolose-6-phosphate phosphatase] gene insertion under the control of native flowering and tuberization promoters. The transgenic potato plants showed increased resilience to heat, photoperiod changes, herbivory, and competition as compared to the control wild type. The addition of excess trehalose to potato plants improved phenotypic stability in unfavorable environments and showed promise for more resilient agriculture. Overall, this exploration emphasizes the adaptability of plants to stress and the need for targeted interventions in suboptimal environments.

Glyphosate resistance among weed species has been an increasing problem in crop production over the past two decades. Glyphosate resistant Erigeron canadensis (horseweed) has been found on multiple continents and 20 U.S. states. It appears that the resistance is of non-target type, with mechanisms of action that have yet to be elucidated at the gene level. For the first portion of the study, I focused on reproducing the original indirect organogenesis protocol but discovered biotype variability can influence regeneration success. An improved indirect organogenesis method was then developed using the previously published methods that could be used across biotypes. A DBI (DeVerna et al. 1984) based solid medium (I Macro/I Micro Murashige and Skoog (MS) salts/vitamins, 3% sucrose, 0.2% gelzan, 2 mg/L kinetin, 0.438 mg/L indole acetic acid (IAA), pH 5.8) called DBIX was statistically significant (p

As humans pursue space travel and nuclear energy, the risk of harm from ionizing radiation increases. On Earth or in space, plants are essential to our personal and environmental health. Plants serve as sentinels, bioremediators and food sources in areas of high ionizing radiation, therefore it is essential to understand how ionizing radiation affects plant biology. This work aimed to understand plant responses to ionizing radiation in the potato chassis and apply that knowledge to generate novel phenotypes for nuclear energy and space applications. The first gamma radiation phytosensor was developed for monitoring at standoff distances greater than three meters. The expression of Ramazzottius varieornatus (tardigrade) ‘Damage suppressor’ protein was found to be ineffective at increasing plant radiotolerance. Lastly, ionizing radiation response was characterized at low doses, revealing a threshold of response in plants between 2 and 4 Gy. This information can be used to create more sensitive radiation phytosensors and additional radioprotection strategies. All of the work here relied on tissue-specific expression patterns, resulting in a final research chapter on the application of artificial intelligence to understanding cell type differences, so that improved engineering strategies can be generated. This work developed the first field-deployable radiation phytosensor and laid the groundwork for many future phytosensors and radiotolerant plant cultivars. Through this work, humanity can pursue a safer relationship with ionizing radiation.

Organisms exist under constantly varying environmental and internal conditions, which necessitate the differential regulation of gene expression. To synthesize proteins, the ribosome translates the information encoded in the nucleotide sequence of an mRNA into the final, functional amino acid sequence. Knockouts of ribosomal proteins lead to lethality. One such protein is the ribosomal protein 6 of the small subunit (eS6/RPS6). We confirmed that the knockout of either one of two eS6 paralogs in Arabidopsis leads to stunted growth and chlorosis. Here, these phenotypes have been further characterized in seedlings by precisely quantifying the ribosome loading of mRNAs as well as by RNA-seq and proteomics. Loss of either RPS6 paralog causes widespread perturbations in chloroplast development, including in photosystem (PS)II and PSI, as well as ribosome biogenesis. Furthermore, we assess whether the phosphorylation status of eS6 plays a role in its regulation. Elimination of eS6 phosphorylation resulted in a mild phenotype with minor changes to the transcriptome, suggesting it is dispensable for most aspects of plant development. To more broadly understand the process of translation we developed a mathematical model that predicts the transcript population and the transcript’s association with ribosomes. Computational techniques have become a cornerstone of modern biology allowing for broad molecular characterization and subsequent development of formal explanations of biological phenomena. In this model we focus on the effect of transcript decapping on the mRNA population and its translational output. As expected, increased decapping reduces overall protein production, partly by reducing ribosomal load, but mainly by shifting the mRNA population from the translationally active, capped state to the inactive, decapped state. Additionally, in transcripts with low half-lives, we find that a significant proportion of the protein production occurs during cotranslational decay. Through these two projects we examine the ability of Arabidopsis to regulate its protein production.

Poplar (Populus sp.) is a promising biofuel feedstock due to advantageous features such as fast growth, the ability to grow on marginal land, and relatively low lignin content. However, there is tremendous variability associated with the composition of biomass. Understanding this variability, especially in lignin, is crucial to developing and implementing financially viable, integrated biorefineries. Although lignin is typically described as being comprised of three primary monolignols (syringyl, guaiacyl, p-hydroxyphenyl), it is a highly irregular biopolymer that can incorporate non-canonical monolignols. It is also connected by a variety of interunit linkages, adding to its complexity. Secondary cell wall formation requires the coordination of many metabolic pathways. Additionally, complex traits such as lignin are highly polygenic. While there are several methods to analyze lignin structure, 2D HSQC NMR is a powerful analytical tool that elucidates many structural traits of lignin simultaneously. This work examined the lignin structure of 409 unique Populus trichocarpa genotypes via HSQC NMR. Twelve lignin phenotypes were subsequently utilized for a genome-wide association study (GWAS) – a powerful approach for identifying loci contributing to natural phenotypic diversity. This deep phenotyping enabled GWAS to identify 756 candidate genes associated with at least one lignin phenotype. Many of these candidate genes have not been previously reported to be associated with lignin or cell wall biosynthesis. These results provide a valuable resource for gaining insights into the molecular mechanisms of lignin biosynthesis and offer new targets for future genetic improvement in poplar.

The benefit of unmanned aircraft systems and image processing methods in agronomic research across numerous crops has been well documented as has the importance of wheat, Triticum aestivum L., on the global food supply. Hence there is great interest in digital solutions applied to aspects of wheat breeding. A major trait of importance to winter wheat breeders in higher latitudes is winter survival, which can result in poor yield and performance if lines do not survive extreme cold. Scoring winter survival is most commonly based on visual score of 0% to 100% with the higher percentage conveying higher winter survival rates. With the increased interest in hybrid wheat lines, it has brought an increased need to screen for hybrid necrotic lines in the field. With both hybrid necrosis and winter kill reducing the stand count of a plot, the advantageous situation arose to be able to investigate digital solutions of measuring wheat stand and their relationship with winter survival and hybrid necrosis. We were able to show that the utilization of multiple vegetative indices and segmentation indices derived from multispectral imagery within the same linear model was able to predict stand with a correlation of r = 0.836 (p < 0.01; flight date 5/18/2020) to visually scored plot stand data. Using unnormalized RGB model utilizing segmentation indices (an index that is used to separate vegetative pixels from background pixels) was able to achieve a prediction with a correlation of r = 0.924 (p < 0.01; flight date 5/18/2020) with the visually scored plot stand data, lending to the potential use of segmentation in conjunction with processes where RGB images are not normalized. In lines exhibiting symptoms of hybrid necrosis a clear clustering pattern could be observed as the season progressed for NDVI values of hybrid necrotic lines compared to non-necrotic lines. Advisor: Stephen Baenziger

Woody plant encroachment is a global threat and has been transitioning grasslands to woody dominance at a biome scale. This threat is present in the Great Plains grassland biome which is currently experiencing grassland biome collapse as the alternative woody biome advances northwest. Nebraska, which contains the most intact temperate grassland in the world, is currently at the front lines of this large-scale transition making this state’s management decisions vital for the remaining grasslands and the species which rely on these ecosystems. In this study, we assess the vulnerability of Greater Prairie Chicken and Tier 1 at-risk species in Nebraska caused by the threat of grassland transition to woody dominance. Chapter one focuses on the threat of the advancing woody biome to Greater Prairie Chicken in Nebraska, utilizing two measurements to quantify risk. Chapter two utilizes participatory Tier 1 at-risk species assessments to assess statewide and regional vulnerability to Tier 1 species listed in Nebraska’s Natural Legacy project caused by the threat of grassland lost to woody dominance. Overall, the findings show that Greater Prairie Chicken, and key at-risk species found in Nebraska have become increasingly vulnerable to this threat. These results highlight the need for adaptive management strategies to mitigate the risk of woody encroachment and subsequent grassland transition to woody dominance. Advisor: Dirac Twidwell

Triticale (Triticosecale) is a multifunctional hybrid cereal crop that adopted the hardiness of rye and wheat's high-yielding and nutritional qualities. Plant breeding programs work to improve the quality and number of varieties available to producers through multiple rounds of evaluation and selection. However, traditional phenotyping methods are labor-intensive, time-consuming, and destructive, creating a phenotyping bottleneck. Remote sensing using unmanned aerial systems has the potential to alleviate this issue and change the evaluation of phenotypes in a breeding. Demand for educational resources to advance public awareness and prepare the workforce has increased with the utilization of more technology in agriculture. Limited research focuses on using UAV-derived vegetation indices to measure biomass in triticale. In addition, agriculture professionals need more education and understanding about the potential benefits and practical implementation of remote sensing technologies in plant breeding contexts. This research aims to (1) Evaluate the potential of UAV-derived vegetation indices to estimate above-ground biomass in triticale and (2) Assess the impact of open educational resources on the change in self-reported and objectively assessed knowledge of phenotype evaluation using high throughput phenotyping. The results of study one show high correlations between biomass and several vegetation indices, indicating that UAV-derived vegetation indices have the potential to be used as an alternative to destructive biomass sampling for phenotyping biomass in triticale. The results of study two show the open educational resource High Throughput Phenotyping in Plant Breeding increases learner overall self-reported knowledge, UAV self-reported knowledge and cross-listed self-reported knowledge. In addition, the lesson increases overall objectively assessed knowledge and cross-listed objectively assessed knowledge. Advisors: Donald Lee and Leah Sandall

Eastern redcedar (Juniperus virginiana L. var. virginiana) is a native species currently invading open areas and grasslands outside of its original range in the United States. I studied the eastern redcedar’s (ERC) invasion patterns in the Lakeside Daisy State Nature Preserve (LDSNP), a short grass prairie located on the Marblehead Peninsula in Ohio, examining the changes in the genetic diversity and structure of the encroaching population. I investigated the relative importance of long-distance dispersal vs. diffusion in the invasion of this short grass prairie by ERC. I used eight microsatellite marker loci and a database of single nucleotide polymorphisms to infer gene flow from external sources vs. within-population recruitment. I found that the older trees in this preserve were less than fifty-years-old, indicating that the population was established between 1970 and 1980. When I grouped trees into five age categories of 10-year increments, we found that the allelic diversity, as indicated by the average number of alleles per locus, increased as the age of the trees decreased. Principal Coordinate Analysis showed two distinct groups of trees in the LDSNP that I investigated further using soil type. Analysis of the population structure of the ERC trees using ADMIXTURE revealed three ancestral clusters in the ERC populations. All ancestral clusters are present in all age groups, suggesting that there is continual input of genetic information from the ancestral clusters. Overall, my findings indicate that ERC encroachment of the LDSNP results from multiple and reiterated gene flow events from the edge of the range through animal-mediated seed dispersal at short and intermediate distances.

Oxygenic photosynthesis supports the majority of life on Earth through the capture of energy from sunlight and the assimilation of CO2 into basic building blocks of cells. Microalgae are fast growing and account for about half of global photosynthesis. In addition, they can be cultivated and their metabolism can be redirected to generate additional useful products ranging from biofuels to pharmaceuticals. However, the efficiency of metabolite production is severely impacted by the slow diffusion of CO2 through water and the high energetic costs of harvesting microalgae from liquid cultures. Microalgae grow in open water, but they also form biofilms that require less energy to harvest. However, the impact of these different growth forms on rates of photosynthesis is poorly understood. The work in this dissertation explores the importance of growth form on photosynthesis by examining CO2 assimilation of the green microalga, Chlamydomonas reinhardtii, within three different states: as a liquid suspension, as a simple filtered two-dimensional artificial biofilm, and within a silica sol-gel encapsulation matrix as an example of a more complex biofilm. The rates of CO2 assimilation were decreased within the simple filtered biofilm and further decreased within the silica sol-gel matrix. The decrease is thought to be due to the diffusional limitations to CO2 imposed by the biofilm forms. Estimated rates of assimilation of CO2 were also calculated from chlorophyll fluorescence values of both biofilms and were more similar to the measured liquid suspension rates, suggesting a persistent energetic imbalance between light and CO2 capture in biofilms. This effort required development of new empirical corrections to correctly assess CO2 exchange rates, and novel approaches to collect data that could be directly compared between the three forms.

Two species of mint, Mentha piperita (peppermint) and Mentha spicata (spearmint), are cultivated across the U.S. Mint is an aromatic herb and perennial crop grown for several aromatic oils, among which menthol and carvone are the primary oils for peppermint and spearmint, respectively. Currently, farmers in the Nebraska Panhandle use different nitrogen (N) rates and various N fertilizers in mint production because there is no information available regarding the optimum rate and source of N fertilizer for mint production in the area. Therefore, optimized N management in mint production could increase aromatic oil yield and quality and reduce greenhouse gas emissions (nitrous oxide; N2O). Nitrous oxide is a major greenhouse gas and the single most important ozone-depleting emission. Increasing N2O emissions from agriculture are linked to soil management and the application of N fertilizers. The objective of this research was to assess the effects of fertilizer N sources (PCU; polymer-coated urea and urea) and rates on dry matter yield and oil concentration in irrigated peppermint and spearmint. Additionally, the experiment aimed to assess the effects of fertilizer N rates and sources on N2O emissions in peppermint production. The experiment was conducted in 2022-2023 at the University of Nebraska Panhandle Research and Extension Center near Scottsbluff, Nebraska. The experimental design was a randomized complete block with four replicates. The main factor was N treatment, which included the control, urea, and PCU surface applied at different rates, resulting in five different soil available N levels (spring soil test N + applied N). In 2022, dry matter yield ranged from 3.33 to 3.98 Mg ha-1 in peppermint and 2.33 to 2.63 Mg ha-1 in spearmint. Only spearmint dry matter yield was significantly affected by N rates and N sources in 2022. In 2023, dry matter yield ranged from 7.56 to 13.28 Mg ha-1 in peppermint and from 10.41 to 13.43 Mg ha-1 in spearmint. Mint dry matter yield was greater in 2023 than in 2022, as 2022 received less N than 2023 and had plant establishment issues. In 2023, greater peppermint dry matter yield was obtained from PCU and urea at 248 kg N ha-1 , while greater spearmint dry matter yield was obtained from 161 kg N ha-1 of soil available N with PCU which showed the best results compared to urea. Oil concentrations in peppermint leaves in the fertilized treatments were greater than in the control but they did not vary by applied N rates or sources, while spearmint oil concentrations did not show any response across N rates and sources. The urea treatments had greater N2O emissions than PCU across all N rates in both years, except for the lowest N rate in 2022. Data reported an incremental trend of N2O emission with an increment of soil available N in the urea treatments but not in the PCU in both years. N2O emission from PCU did not differ from control in both years. The results suggest that the N application can be optimized to improve the performance of sustainable mint production using advanced fertilizer technology such as PCU. However, more site-year data would be necessary to determine the optimum N rates. Advisor: Bijesh Maharjan

The nucleus is a dynamic organelle whose nuclear envelope (NE) is composed of a double membrane with a distinct but continuous outer nuclear membrane (ONM) and inner nuclear membrane (INM). Linker of the nucleoskeleton and cytoskeleton (LINC) complexes are located at the nuclear envelope and are composed of Klarsicht/ANC-1/Syne Homology (KASH) ONM proteins and Sad1/UNC-84 (SUN) INM proteins. SUN and KASH proteins interact in the NE lumen, forming a bridge between the nucleoplasm and cytoplasm. While plants do not encode homologs of animal KASH proteins, functional analogs have been identified. The Arabidopsis thaliana WPP domain-interacting proteins (WIPs) were the first plant KASH proteins identified. These proteins interact with WPP domain-interacting tail-anchored (WIT) proteins at the ONM, and with Arabidopsis SUN proteins in the INM. Together, a LINC complex comprised of SUN, WIP, and WIT is necessary for proper vegetative nuclear movement and morphology in pollen tubes.In Arabidopsis pollen tubes, the vegetative nucleus (VN) precedes the sperm cells (SCs) and maintains a fixed distance from the pollen tube tip during growth. In pollen tubes of wip and wit mutants, the SCs precede the VN during pollen tube growth and the fixed VN distance from the tip is lost. Pollen tubes frequently fail to burst upon reception at the ovule, leading to a significant fertility defect. In vitro-induction of reactive oxygen species (ROS) has been shown previously to lead to pollen tube growth arrest and rupture in a Ca2+-dependent manner. I hypothesized that the WIT-WIP-SUN LINC complex is required for ROS-induced pollen tube rupture. I report a decrease in Ca2+-dependent ROS-induced pollen tube rupture in both wip and wit mutants. The ROS hyposensitivity correlates with an increased distance of the VN from the pollen tube tip. In addition, I report the first evidence of nuclear Ca2+ fluctuations in the VN and show that the patterns during growth and ROS-mediated pollen tube burst are altered in the mutants. The proximity of the VN to the pollen tube tip is required for WT-like response to exogenous ROS and nuclear Ca2+ fluctuations.In addition to WIPs, four additional KASH proteins were bioinformatically identified and named SUN domain-interacting nuclear envelope (SINE) proteins. SINE1 and SINE2 are paralogous proteins that have been shown to function during stomatal dynamics induced by light-dark transitions and ABA. ABA is a plant hormone that induces stomatal closure in response to stress, involving the activation of guard cell anion channels, reactive oxygen species (ROS) production, cytoplasmic calcium (Ca2+) oscillations, and filamentous actin (F-actin) reorganization. I show that ABA-induced ROS production is not perturbed in sine1-1 and sine2-1 mutants. However, ABA-induced cytoplasmic Ca2+ oscillations are diminished in sine2-1 but not sine1-1, and this defect can be rescued by both exogenous Ca2+ and F-actin depolymerization. ABA-induced cytoplasmic Ca2+ oscillations are also diminished in sine1-1 sine2-1 and this defect can be rescued by exogenous Ca2+, but not F-actin depolymerization. I show the first evidence for nuclear Ca2+ oscillations during ABA-induced stomatal closure, which are disrupted in sine2-1.Loss of SINE1 and SINE2 has been shown to impair F-actin reorganization during ABA-induced stomatal closure, in which SINE1 is involved in actin polymerization and/or stabilization and SINE2 is involved in actin depolymerization. To further elucidate the role SINE1 and SINE2 play during actin reorganization during ABA-induced stomatal closure, I generated double mutants with SINE1 and SINE2 and the two known actin-remodeling factors SCAB1 and the ARP2/3 complex and observed actin reorganization during stomatal closure. I demonstrate that ABA-induced actin reorganization is impaired in sine1-1 sine2-1, similar to sine2-1. I demonstrate that SINE1 and the ARP2/3 complex function in the same pathway during ABA-induced stomatal closure, while SINE2 and the ARP2/3 complex play opposite roles. The additional loss of SCAB1 can partially rescue the actin reorganization defect observed in sine1-1, but not sine2-1. Additionally, loss of SINE1 and the ARP2/3 complex impairs shoot biomass and lateral root development while absence of SINE2 renders trichome development independent of the ARP2/3 complex.Plants have an alternating two-staged life cycle between the haploid gametophyte generation and the diploid sporophyte generation. Flowering plants generate male and female multi-cellular haploid gametophytes for sexual reproduction. In these gametophytes, haploid cells undergo two to three rounds of post-meiotic mitosis, to form gametes and the accessory cells required for reproduction. I show that SINE3, which had not been functionally characterized, plays a role in both male and female gametogenesis. Loss of SINE3 results in a female-derived fertility defect, with sine3 mutant ovules arresting at developmental stage FG1 prior to mitosis. Pollen viability is also significantly reduced in sine3 mutants, as a result of pollen microspores arresting prior to pollen mitosis I. In addition, I demonstrate that sine3 mutants have a minor male meiosis defect, with some tetrads containing more than four spores of different sizes. I show that SINE3 is expressed at all stages of male and female gametophyte development at the nuclear envelope, and that this localization is dependent on its C-terminal KASH domain.The work presented here expands our knowledge on the functions of plant KASH proteins in various plant cellular processes, such as male fertility, stomatal dynamics, and male and female gametogenesis.

The intensive management associated with many annual crops often includes recurring tillage, fertilization, and pesticide applications, which contribute to environmental concerns such as water pollution and soil erosion. Intermediate wheatgrass (Thinopyrum intermedium), recognized under the trade name Kernza®, is a perennial grass that can be managed to produce grain and biomass while providing desired environmental benefits such as soil conservation and nutrient cycling. There has been limited research on best management practices and crop productivity for this alternative dual-use crop in Nebraska. A field experiment was conducted beginning in 2021 to assess nitrogen (N), phosphorus (P), and potassium (K) management practices for intermediate wheatgrass (IWG) grain and forage production and how it changes over the years of the stand. Additionally, a meta-analysis of 16 independent studies was conducted to evaluate the impact of nitrogen rates across sites and years on intermediate wheatgrass productivity. The meta-analysis found limited effects on grain yield in year 1 but optimal rates ranging from 51-150 kg N ha-1 in later years. In our field experiment for years 1 and 2, N, P, and K rates did not impact yields, while second year yields were much lower than the first year, likely due to dry spring conditions during anthesis. Our findings highlight the importance of strategizing the fertilization practices across the intermediate wheatgrass stand years in order to maximize grain and forage production. Moreover, IWG demonstrated its potential as a low-input, alternative, and dual-use cash crop for Nebraska producers. Advisor: Andrea D. Basche

Gravity is among the most critical of environmental cues in shaping plant growth;however, its ubiquity on Earth limits available options in the study of the plant gravityresponse. NASA has circumvented this obstacle by hosting experiments in themicrogravity environment of the International Space Station (ISS). Gene expression datagleaned from RNA-seq and microarray analyses of Arabidopsis seedlings grown aboardthe ISS has provided a wealth of information regarding how plants respond to this uniquegravity condition. A meta-analysis of these datasets intersected with one of terrestrialplants exposed to an alternate gravity stimulus - a new gravity vector – has identifiedseveral novel components in the gravity signaling pathway. Of particular interest in theanalysis were transcription factors, for their role in regulating downstream expressionpatterns. Two transcription factors were identified at this fundamental level of theArabidopsis gravity response: ERF104, which is upregulated on earth in response to anew gravity vector and downregulated in microgravity, and IQD21, which displays theinverse expression pattern. A third gene, CIB1, was shown to be upregulated in bothscenarios. Phenotypic characterizations of mutant lines of each of these genes showsignificant gravity sensitivity in the root tip and/or inflorescence stem, confirming theirgravitropic role. Fusion constructs of these transcription factors joining each with a Human Influenza Hemagglutinin (HA) tag were generated, placed under the control of aconstitutive promoter, and expressed in each gene’s respective Arabidopsis T-DNAmutant line. These lines are enabling chromatin immunoprecipitation followed bysequencing (ChIP-seq) to identify binding sites of each transcription factor in theArabidopsis genome, and consequently to infer their downstream targets. This work wasdone in tandem with qPCR analyses targeting the expression patterns of each protein inresponse to gravistimulation. Together, this research will help to contextualize and clarifythe gravity response pathway, as well as shed light on the complex web of interconnectedsignaling events it entails.

Glycosyltransferases (GTs) are important proteins that are widely distributed in both prokaryotes and eukaryotes and play a crucial role in the biosynthesis of carbohydrates and glycoconjugates. They catalyze the formation of specific glycosidic linkages by transferring sugar moieties from activated sugars to a variety of biomolecules such as carbohydrates, lipids, proteins, or water. Progress in functional genomics technologies, such as DNA sequencing and proteomics, has allowed the identification of a large number of GT genes in several species. In general, functional genomics approaches, which include genomics, genetics, proteomics, and biochemistry, are used to determine the function of a gene (i.e., GTs). In biochemical approaches, the most direct way to assign a function to a gene is through direct testing of the enzyme activity of its product (carbohydrate) in vitro. However, in contrast to genomics/proteomics approaches, the biochemical approaches are the most difficult to adapt to high-throughput screening. The reason is that biochemical approaches require the use of isolated/purified proteins, which is labor-intensive and prone to the formation of undesired products resulting from background enzyme activity. Therefore, there is a need for the development of protein-based in vitro high-throughput platforms for determination of biochemical functions of proteins, including GTs, and their interactions with other proteins. To be advantageous, a protein-based high-throughput platform should have the following characteristics: i) the platform can be adapted to all GTs and synthases, ii) the detection method should be sensitive enough to demonstrate the formation of GT products, and iii) the platform should be simple and easy to implement or accessible to any laboratory. This work describes the development of a novel platform for screening of enzyme activities of GTs in vitro. It is called the in vitro GT-array (i-GTray) platform. This platform uses an in vitro cell-free expression system to produce tagged proteins directly from plasmid DNA and capture the tagged proteins on a solid surface, such as microplate wells, using a capture antibody (anti-tag antibody). Thus, i-GTray combines protein synthesis and purification with post-assay desalting paper spray-mass spectrometry (DPS-MS) analysis. DPS-MS has the capability to detect as low as 50 fmol amounts of transferase products.As a proof-of-concept, the i-GTray platform was used to investigate the activity of 21 putative fucosyltransferases (FUTs) members of the GT37 family (CAZy database) using five different acceptor substrates representing fucosylated plant CW polymers, such as xyloglucan (XyG), arabinogalactan-proteins (AGPs), and pectin (rhamnogalacturonan I and II (RG-I and RG-II)). This screening consisted in 288 assays (four microplates) and resulted in the identification of five rice XyG-FUTs, four rice AGP-FUTs, three Arabidopsis RG-I-FUTs, and one Arabidopsis RG-II-FUT. However, none of the 15-rice putative FUTs acted on RG-I and RG-II acceptors used in this study. The i-GTray platform was also used to investigate the specificity of protein interactions among rice GTs belonging to the GT43 and GT47 families, and the results obtained corroborated BiFC data. Data from both i-GTray and BiFC are in agreement and demonstrated the assembly of at least six OsGT43/OsGT47 complexes that represent the central core complexes of three xylan synthase complexes (OsXSCs).

Masting is defined as highly synchronized seed production within a population that varies in intensity among years. Reproductive output of masting species and the occurrence of years with high seed production are thought to be influenced by climate and resource availability. In oaks, spring weather seems to be particularly influential to seed production. However, there is also significant variation in seed production among individuals in the same population. The goal of this study was to examine if individual variation in seed production of Quercus montana (chestnut oak) was explained by differences in pollen availability. Phenological synchrony and conspecific density were examined as indicators of pollen availability. I also asked if these variables were influenced by differences in microclimate related to topography and spring weather. The study included 36 Q. montana individuals from populations in Vinton Furnace Experimental Forest and Zaleski State Forest in southeastern Ohio. For each individual, microclimate was quantified by collecting data on elevation, slope, aspect, air temperature, soil temperature, and humidity during spring of 2022 and 2023. Observations on individual flower development were made from March to May of 2022 and 2023, and relative density of conspecifics within 50 m was also quantified. Results showed that individuals at higher elevations and on more south-facing slopes experienced warmer and drier microclimate conditions during spring, but these conditions did not significantly influence phenological synchrony in either year. Based on seed production data from 2022, there was not a significant relationship between either phenological synchrony nor conspecific density and individual seed production, however there was a significant positive relationship between individual dry weight of collected catkins and number of acorns produced. Analysis of long-term seed production collected since 2000 also revealed a positive relationship between elevation and seed production and an insignificant effect of conspecific density. Overall, my results suggest that there is some reproductive benefit related to microclimate, as individuals with consistently high or frequent seed production are at higher elevations and experience warmer microclimate conditions. However, this benefit does not seem to be related to pollen limitation. Intraspecific variation in seed production was influenced by variation in total catkin biomass, and future work could examine potential links between microclimate, resource investment in flowers, and pollination efficiency.

A green roof provides different benefits in an urban environment, like habitat, stormwater management, reduced energy usage, aesthetic values, and carbon storage. To understand the carbon storage capacity of green roofs, it is important to understand the amount of CO2 released (efflux) and taken up by the green roof. So, I measured and compared the above and belowground biomass, soil CO2 efflux (respiration), and associated soil parameters between the green roof and the prairie site to understand how environmental variables like soil temperature and moisture on the green roof influence CO2 efflux. The green roof substrate was significantly warmer than the prairie soil, and CO2 efflux from the green roof was less sensitive to temperature (i.e., had lower Q10) than the prairie site. I hypothesize that the porosity of the green roof substrate, which accounts for its lower water-holding capacity and volumetric moisture content, lowered the potential evapotranspiration and caused higher temperature and greater variation in temperature on the green roof. The green roof also had comparatively higher variation in substrate temperature, consistent with greater variation in CO2 efflux. The differences in the plant activity led to the major differences in the CO2 efflux between the green roof and the prairie site. Higher above and belowground (root biomass) on the prairie site led to higher total CO2 efflux on the prairie site as higher aboveground biomass can lead to increased photosynthates for the roots and higher root exudates along with the higher litter input, all of which can increase autotrophic and heterotrophic respiration. However, after accounting for the differences in root biomass on soil CO2 efflux (i.e., dividing efflux by root biomass), the green roof had higher efflux than the prairie sites, indicating a higher contribution of heterotrophic sources to CO2 efflux at the green roof. Since the green roof at Schoonover Center is unique due to its location, climate, aspect, and species composition, the result might not be similar to other green roofs. However, a long-term study of the trends of soil organic matter concentration over the years, along with measurements of parameters like CO2 efflux and net primary productivity, can help determine whether the green roof at Schoonover Center can store carbon.