Browsing by Author "Bush, Daniel, committee member"

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Open Access
A new woody perspective on copper homeostasis: systemic copper transport and distribution, effect of copper on lignification, and water transport in hybrid poplar
(Colorado State University. Libraries, 2022) Hunter, Cameron Ross, author; Pilon, Marinus, advisor; Gleason, Sean, advisor; Pilon-Smits, Elizabeth, committee member; Argueso, Cristiana, committee member; Bush, Daniel, committee member
Copper (Cu) is an essential micronutrient for plants. Chapter 1, as background for this dissertation, reviews the functions and homeostasis of Cu. We know at the cellular level how Cu is delivered to target proteins in the chloroplasts, thus explaining in a large part why Cu deficient plants have reduced photosynthetic capacity. However, Cu is also a cofactor of lignin polymerization enzymes that affect cell wall and xylem structures required for water and mineral transport. How Cu deficiency affects water transport, mineral nutrition, and photosynthesis at a whole plant level is underexplored. To address this knowledge gap, we used hybrid white poplar as a model. In chapter 2, a stable isotope method to trace Cu movement in poplar tissues was coupled with analysis of photosynthesis and stomatal conductance. Upon resupply of Cu, priority targets identified were stems and younger leaves which recovered quickly and was associated with higher stomatal conductance. In chapter 3, the effect of Cu deficiency on the elemental composition of leaves and stems of different age were analyzed. Interestingly, tissue type and age, as well as Cu deficiency, were found to all significantly affect within-plant nutrient partitioning patterns. In chapter 4, the effects of Cu deficiency on cell wall chemical composition and water transport traits were determined. Although Cu deficiency strongly affected cell wall chemistry, it did not significantly impact hydraulic capacity nor the density and size of xylem vessels in stems. However, Cu deficiency resulted in markedly stiffer mesophyll cell walls, possibly arising from changes to cell wall chemistry or structure. Together, these results, as discussed in chapter 5, indicate that although xylem lignification was adversely affected by Cu deficiency, the water transporting vessels remained largely unaffected, thus allowing efficient recovery. This work opens new avenues to explore the effects of plant nutrition on whole-plant physiology and function.
Open Access
Analysis of genome-wide targets of Arabidopsis signal responsive 1 (AtSR1) transcription factor and its transcript stability in response to stress
(Colorado State University. Libraries, 2017) Abdel-Hameed, Amira, author; Reddy, A. S. N., advisor; Bush, Daniel, committee member; Leach, Jan, committee member; Abdel-Ghany, Salah, committee member
Abiotic and biotic stresses cause significant yield losses in all crops. Acquisition of stress tolerance in plants requires rapid reprogramming of gene expression. SR1/CAMTA3, a member of signal responsive transcription factors (TFs), functions both as a positive and a negative regulator of biotic stress responses and as a positive regulator of cold stress-induced gene expression. Using high throughput RNA-seq, we identified ~3000 SR1-regulated genes. Promoters of about 60% of the differentially expressed genes have a known DNA binding site for SR1, suggesting that they are likely direct targets. Gene ontology analysis of SR1-regulated genes confirmed previously known functions of SR1 and uncovered a potential role for this TF in salt stress. Our results showed that SR1 mutant is more tolerant to salt stress than the wild type and complemented line. Improved tolerance of sr1 seedlings to salt is accompanied with the induction of salt-responsive genes. Furthermore, ChIP-PCR results showed that SR1 binds to promoters of several salt-responsive genes. These results suggest that SR1 acts as a negative regulator of salt tolerance by directly repressing the expression of salt-responsive genes. Overall, this study identified SR1-regulated genes globally and uncovered a previously uncharacterized role for SR1 in salt stress response. Soil salinity, one of the most prevalent environmental stresses, causes enormous losses in global crop yields every year. Therefore, it is imperative to generate salt tolerant cultivars. To achieve this goal, it is essential to understand the mechanisms by which plants respond to and cope with salt stress. Stress-induced reprogramming of gene expression at multiple levels contributes to the survival of plants under adverse environmental conditions. The control of mRNA stability is one of the post-transcriptional mechanisms that is highly regulated under stress conditions leading to changes in expression pattern of many genes. In this study, we show that salt stress increases the level of SR1 mRNA, by enhancing its stability. Multiple lines of evidence indicate that ROS generated by NADPH oxidase activity mediate salt-induced SR1 transcript stability. Furthermore, cycloheximide (CHX), a protein synthesis inhibitor, also increased SR1 mRNA stability, albeit to a higher level than in the presence of salt, suggesting a role for one or more labile proteins in SR1 mRNA turnover. Similar to salt, ROS generated by NADPH oxidase is also involved in CHX-induced SR1 mRNA accumulation. To gain further insights into mechanisms involved in saltand CHX-induced SR1 stability, the roles of different mRNA degradation pathways were examined in mutants that are impaired in either nonsense-mediated decay (NMD) or mRNA decapping pathways. These studies have revealed that neither the NMD pathway nor the decapping of SR1 mRNA is required for its decay. However, decapping activity is required for saltand CHXaccumulation of SR1 mRNA. To identify any specific regions within the open reading frame of the SR1 transcript (~3 kb) that are responsible for the salt-induced accumulation of SR1 mRNA, we generated transgenic lines expressing several truncated versions of the SR1 coding region in the sr1 mutant background. Then, we analyzed accumulation of each version in response to salt stress and CHX. Interestingly, we identified a 500 nts region in the 3' end of the SR1 coding sequence to be required for both saltand CHX-induced stability of SR1 mRNA. Potential mechanisms by which this region confers SR1 transcript stability in response to salt and CHX are discussed.
Open Access
Biology, comparative genomics and molecular diagnostics of Xanthomonas species infecting rice and corn
(Colorado State University. Libraries, 2017) Lang, Jillian M., author; Leach, Jan E., advisor; Bush, Daniel, committee member; Reddy, Anireddy, committee member; Verdier, Valérie, committee member
Emerging bacterial diseases on staple and economically important crops can pose critical threats to food security. Accurate identification of bacterial plant pathogens is the foundation of effective management for growers. This work advances the application of genomics to identify and characterize bacterial plant pathogens in the genus Xanthomonas that can cause destructive diseases on most agricultural crops, including rice and corn. In this thesis, taxonomy, host range, disease phenotypes and basic biology of the following pathogens were established: X. oryzae pv. oryzae, X. o. pv. oryzicola, X. o. pv. leersiae and X. vasicola pv. vasculorum. X. o. pv. oryzae and X. o. pv. oryzicola infect rice and cause bacterial blight and bacterial leaf streak, respectively. X. o. pv. leersiae infects cutgrass (Leersia sp.), weedy grasses that can serve as alternative hosts to X. oryzae and are endemic in all rice growing regions. X. vasicola pv. vasculorum was identified as the causal agent of bacterial leaf streak of corn, an emerging and now wide-spread disease in the United States, that was reported for the first time in 2017. This work established that X. vasicola pv. vasculorum can also infect sorghum and sugarcane and that the US strain is 99% similar to strains isolated over 20 years ago in S. Africa. To develop robust molecular diagnostic tools for these pathogens, unique features needed to be first identified. Using comparative genomics that included closely related bacteria and distant relatives, PCR-based diagnostic tools were developed, then validated using isolated cultures and field grown plant materials. Comparative genomics also contributed to elucidation of the taxonomy and phylogeny of X. o. pv. leersiae and X. v. pv. vasculorum. Characterization of X. o. pv. leersiae revealed adaptations to both the weedy grass hosts and rice. These features include virulence proteins that target homologous host genes (transcription activator like effectors, TALEs) to influence host gene expression. I conclude that X. oryzae is a complex that includes X. oryzae pv. oryzae, X. o. pv. oryzicola and X. o. pv. leersiae and that this complex can provide a unique window into pathogen evolution. By better understanding how pathogens adapt to their environments including new hosts, growers can manage surrounding ecosystems more effectively to minimize yield losses and therefore, contribute to food security.
Embargo
Characterization and insights into the molecular mechanism of cytokinin-induced priming of plant defenses
(Colorado State University. Libraries, 2023) McIntyre, Kathryn, author; Argueso, Cristiana, advisor; Bush, Daniel, committee member; Leach, Jan, committee member; Stewart, Jane, committee member; Reddy, Anireddy, committee member
Plants have developed several mechanisms to cope with pathogenic challenges. One of these mechanisms, known as defense priming can be effective at reducing susceptibility to pathogens. Compared to unprimed plants, the immune response from primed plants, upon pathogen attack, is much stronger. This mechanism of induced disease resistance can be initiated by biological and chemical agents. The major benefit of priming is the induction of a high level of protection with considerably low fitness costs making it an attractive disease management strategy to preserve agricultural output. Recent research has demonstrated that the plant hormone cytokinin (CK) has a priming effect against biotrophic pathogens, a phenomenon referred to here as cytokinin-induced priming (CIP). This dissertation aims to gain further understanding of CIP against the hemibiotrophic bacterial pathogens Pseudomonas syringae pv. tomato (Pst) and Pseudomonas syringae pv. maculicola (Psm) in Arabidopsis thaliana (Arabidopsis) and Brassica napus, respectively as well as the necrotrophic fungal pathogen Botrytis cinerea in Arabidopsis. Chapter 2 focuses on characterizing CIP as a true priming agent by investigating the timeframe in which CIP is most effective at reducing susceptibility to Pst and Psm in both Arabidopsis and its closely related relative, B. napus and the impacts on plant growth due to CIP in these pathosystems. Moreover, we discovered that other known priming agents depend on endogenous CK signaling suggesting CK-mediated processes are involved in the priming of defense responses. The role of CK in primed defenses against B. cinerea is explored in chapter 3 where CIP is demonstrated to reduce necrotic lesion size caused by B. cinerea in a manner dependent on the JA-mediated defenses and partially on SA-mediated defenses. Transcriptome analysis revealed that during the priming stage, CK prepares the plants for pathogenic challenge through the accumulation of cellular components needed for translation and metabolites utilized for energy production and defense. Following B. cinerea inoculation, CIP suppresses defense while increasing photosynthetic-related processes. In the final chapter, molecular mechanisms are explored during CIP against Pst. Through transcriptome changes, priming by CK potentiates gene expression associated with systemic induction of defense, also known as systemic acquired resistance (SAR), following Pst challenge. Using this information, it is demonstrated that CK treatment can also induce SAR and that the known SAR inducer, L-pipecolic acid, is dependent on endogenous CK signaling. Due to the previously identified relationship between CK and source-sink relationships, amino acid transport was demonstrated to have a role in both CIP and CK-induced SAR. New agricultural practices that mitigate crop loss due to plant diseases are beneficial in terms of sustainability and economic costs. The use of CK as a priming agent offers an avenue for a new disease management strategy in that CIP protects plants against a broad range of pathogens with minimal effects on plant growth. The molecular mechanisms underlying CIP discovered here offers new insights into the relationship between plant metabolism and defense, where its exploitation could be used to create disease protection strategies.
Open Access
Characterization of a synthetic signal transduction system
(Colorado State University. Libraries, 2012) Albrecht, Tessa, author; Medford, June, advisor; Bush, Daniel, committee member; Pilon-Smits, Elizabeth, committee member; Leach, Jan, committee member
The Medford laboratory has developed a synthetic signal transduction system linking exogenous perception of a particular ligand to a transcriptional response. One application of this system is to produce plants that sense and respond to a specific ligand. The system was designed based on evolutionary conservation of histidine kinase signaling and uses bacterial components adapted to function in plants. The synthetic signaling system is responsive to extracellular ligand perception by a wild-type or modified ribose binding protein (RBP) scaffold. Upon ligand binding, RBP binds and activates a synthetic fusion histidine kinase made from the extracellular portion of the bacterial chemotactic receptor Trg and the cytoplasmic portion of the bacterial phosphate sensor PhoR. Activated Trg-PhoR transmits a phosphate signal to the bacterial response regulator PhoB. Upon phosphorylation PhoB translocates into the nucleus of a plant cell and activates transcription of the response gene(s). In addition to receiving a phosphate signal from Trg-PhoR, PhoB can be activated by exogenous cytokinin application suggesting that components of the cytokinin signaling pathway can interact with PhoB. Elimination or reduction of the interaction with cytokinin signaling components allows production of a more reliable signaling system. One goal of the following work was to reduce the interaction of PhoB with endogenous cytokinin signaling components. I attempted to identify a mutant form of PhoB that does not interact with cytokinin signaling components yet maintains function with the synthetic signaling system. I screened six different rationally selected PhoB mutants in plants for reduced response to exogenous cytokinin application. I concluded that a different approach will be needed to successfully reduce interaction with cytokinin signaling components. Another goal of this work was the identification cytokinin signaling components that interact with PhoB, possibly revealing a means to eliminate the interaction. I attempted to functionally express selected cytokinin signaling components in a bacterial testing system. After several failed cloning strategies, I conclude that the cytokinin sensor histidine kinase, AHK4, may be toxic and/or unstable in bacteria and expression of alternative genes will be needed to identify cytokinin signaling components that interact with PhoB. Additional work described here includes the independent testing of two computationally designed RBPs; one reported to bind the environmental pollutant methyl tert-butyl ether and the other reported to bind the explosive trinitrotoluene, for ligand dependent activation of the synthetic signaling system. These results show that the computationally designed RBPs do not function in a reliable manner and lead to the production of a detector plant using wild-type RBP to activate the synthetic signaling system that enables further analysis of the system components in plants.
Open Access
Characterizing the role of plant hormones during plant development and plant immunity in Arabidopsis thaliana and Solanum lycopersicum cv. Micro-Tom
(Colorado State University. Libraries, 2022) Berry, Hannah Marie, author; Argueso, Cristiana, advisor; Bush, Daniel, committee member; Bedinger, Patricia, committee member; Stasevich, Timothy, committee member
Plant hormones are major regulators of plant growth, development, and responses to biotic and abiotic stressors. Constitutive activation of immunity is commonly associated with stunted plant growth. This phenomenon, called the growth defense tradeoff, was previously thought to result from limitations in metabolic processes, where resources were redirected from plant growth toward energetically costly defense responses. However, recent studies have shown that metabolic limitations are not solely responsible for the growth defense tradeoff, and that growth and defense can be uncoupled, resulting in plants with increased immunity without compromising plant yield. While the effects on plant growth have been widely characterized in the context of constitutive immunity, developmental impacts such as changes to plant architecture, reproductive development, and leaf morphology, have been studied to a lesser extent. Cytokinin is one of nine major plant hormone families and its role in plant growth, meristematic maintenance, cell division, and senescence are widely known. In conjunction with salicylic acid (SA), a primarily defense related hormone, cytokinin acts to promote SA-dependent defense responses, thus demonstrating a role for cytokinin in plant immunity. Perception of cytokinin initiates a two-component signaling phosphorelay leading to the activation of downstream transcription factors to induce the transcription of cytokinin-responsive genes. One group of these transcription factors is the CYTOKININ RESPONSE FACTORS (CRFs), which is found in all land plant species. In Chapter 2, I show that CRFs are negative regulators of plant growth and positively regulate plant defense responses. To further elucidate the role of CRFs in growth and immunity, I quantified growth and development in crosses between constitutive immunity mutants with elevated SA and CRF overexpressing (hereafter CRFox) lines. Here, these data show that CRFox enhances the growth restriction phenotypes previously characterized in the constitutive immunity mutants. I propose a model with CRF5 at the intersection of CK and SA crosstalk, acting as a regulator of the growth defense tradeoff. How constitutive immunity alters plant development as well as plant growth is not well understood. In Chapter 3, I characterize changes in plant architecture in constitutive immunity mutants SUPPRESSOR OF NPR1-1 (NONEXPRESSER OF PATHOGENESIS RELATED GENES 1), CONSTITUTIVE 1 (snc1) and CONSTITUTIVE EXPRESSION OF PR GENES 5 (cpr5). Both snc1 and cpr5 have elevated levels of endogenous SA and elevated disease resistance. While the reduced growth phenotypes (measured as biomass) of these mutants have been characterized, phyllotaxy has not previously been quantified. Phyllotaxy describes the consistent arrangement or pattern of consecutive organs around a central point. Arabidopsis has a spiral phyllotactic pattern where each consecutive organ is separated by approximately 137.5°. Phyllotactic analysis of shoot apical meristems (SAMs) using scanning electron microscopy and the arrangement of siliques on inflorescence stems in snc1 mutants showed a change in phyllotactic divergence angle originating from reduced shoot apical meristem size and increased plastochron ratio. The plastochron describes the amount of time between organ initiations but can be shown as a ratio when the edge of two consecutive inflorescence primordia to the SAM center is quantified using imaging software. To mimic the phenotypes of constitutive immunity, I inoculated wild type Arabidopsis Col-0 plants with high concentrations of Pseudomonas syringae pv. tomato (Pst) strains: (1) Pst hrcC-, which is lacks the type-III secretion system necessary to introduce bacterial effectors into the plant cell but initiates plant basal immune responses, thus inducing pattern triggered immunity (PTI); (2) Pst DC3000, which causes plant disease via introduction of bacterial effector proteins into the plant cell to change plant metabolism and dampen plant immune responses causing effector triggered susceptibility (ETS); and (3) Pst ArvRpm1 where the bacterial effector protein Rpm1 is recognized by the plant, initiating a high level of defense responses called effector triggered immunity (ETI). Only multiple, concentrated inoculations of Pst DC3000 were able to induce changes to silique phyllotactic patterns. Notably, the SAM was unaltered after Pst DC3000 inoculations, demonstrating, that phyllotactic patterns originating at the meristem are very robust upon infection, and this change in silique patterning was determined to be a result of post-meristematic stem torsion. I conclude that elevated SA throughout the plant life in the constitutive immunity mutants shows a role for SA in regulating meristematic maintenance and/or patterning, but elevated SA after pathogen attack is not sufficient to overcome the tight regulation of the meristem. Arabidopsis has been a primary source of knowledge for elucidating hormone crosstalk during plant development and immunity. However, Arabidopsis cannot inform us about the roles of hormones during fleshy fruit development. Tomatoes are an important agricultural crop species, have a fully sequenced genome, and many genetic resources are available, making tomatoes a model crop species. In my last research chapter, I quantified plant hormones in Solanum lycopersicum cv. Micro-Tom in above- and below-ground plant tissues at four stages of plant development. I selected plant developmental stages based on easily definable traits: the seedling stage was defined as the presence of cotyledons before the emergence of true leaves; the young developmental stage was characterized by the presence of four true leaves before the transition to flowering; the adult or flowering stage was determined by the opening of the first flower; and the fruiting stage was identified by fruit set and fully expanded, breaker, and ripe fruit development stages. While the data collected in this chapter is primarily descriptive, we showed that a single extraction protocol could be used to extract and quantify 18 plant hormones representing 5 of the 9 major hormone families in multiple tissue types including roots, leaves, and fruits. Plant hormone data were integrated into botanical illustrations to create the Plant Hormone Atlas, which was presented at the Art Lab Fort Collins, in Fort Collins, CO.
Open Access
Gene expression regulation by a stress-responsive transcription factor in rice seedlings
(Colorado State University. Libraries, 2019) Williams, Seré., author; Reddy, A. S. N., advisor; Leach, Jan, committee member; Bush, Daniel, committee member
Stress physiology is an inherently complex field. As plants cannot leave their environment when it becomes unfavorable, they have developed multiple mechanisms to cope with stresses. Many of these are unique to plants compared to mobile organisms. Plant stress physiology is of interest not only for this reason, but because the human population relies on agriculture for food. Additionally, our ecosystem relies on plants as primary producers as an integral component of life on earth. Plant stress physiology at the molecular level involves a symphony of signaling cascades that reshape cell physiology and communicate the stress signal to the whole plant and even nearby organisms. Over the last thirty years, enormous progress has been made to identify key genes, hormones, and signaling pathways that are involved in plant stress responses. To this end, we have yet to understand a cohesive picture of how plants respond to a combination of stresses. Given the variety of biotic stresses from bacteria, fungi, viruses, nematodes, and herbivores and their interaction with abiotic stresses including environmental extremes and resource availability, continued efforts are needed to understand the molecular nuances of plant stress responses. Not only are stresses variable and unique, plants have evolved to thrive in specific habitats, thereby developing unique strategies to cope with local environments. For example, rice grows well in flooded soils which would induce a stress-response in typical, non-aquatic organisms. Therefore, stress response will need to be decoded at the level of the organism. The goal of this work is to better elucidate stress response in rice. Specifically, I have looked at the influence of a transcription factor, SIGNAL RESPONSIVE 1 (OsSR1), that is regulated by Ca2+/CaM and known to be a dynamic regulator in a myriad of stresses in Arabidopsis. I have generated complemented lines of Ossr1 mutant and OsSR1 overexpressor transgenic rice lines. When compared with WT and mutant lines, these lines showed a range of OsSR1 expression. These lines will be of great help in deciphering the action of this transcription factor. Homozygous SR1 complemented and overexpressor lines along with WT and Ossr1 mutant will be used in future studies to better understand the action of SR1 in stress response in rice. Additionally, I performed a factorial global gene expression analysis using RNA-seq with WT and Ossr1 lines at the seedling stage in control and drought conditions, which will serve as a breeding ground for hypothesis generation and testing in future studies. Significant differentially expressed (DE) genes show down-regulation of genes encoding serine threonine-protein kinase receptor (SRK)-receptors, kinases, TCP family transcription factor, cytokinin-modifying enzyme and up-regulation of aquaporin, sucrose synthase, G-protein-related, and ferredoxin-nitrate reductase in the mutant when compared to WT. In response to polyethylene glycol (PEG)-induced drought stress, the mutant up-regulated transcription factors (homeobox [HOX]- containing TFs, WRKY, and DIVARICATA), signaling proteins (protein phosphatases), late embryogenesis abundant protein 1 (LEA1), nodulin-related genes, and senescence-associated gene 21 (SAG21), while down-regulating a CaM-dependent protein kinase, efflux transporters, peroxidases, aquaporins, and disease-related genes including Pathogenesis-related protein PRB1-2, disease resistance protein RPS2, and NB-ARC domain-containing protein. Lastly, significant DE genes in the WT illuminate how this important crop plant responds when exposed to PEG-induced drought. Drought induced the expression of MAPKKKs, ethylene-responsive transcription factors (ERFs), HOX TFs, as well as zinc-finger proteins and protein phosphatase 2Cs. In drought, WT down-regulated glycol-lipid transfer proteins, aquaporins, and salt stress-induced proteins. Gene ontology (GO) analysis of significant DE genes showed enrichment of GO terms related to membranes, oxidative stress, response to stimulus, and transcription regulation in both the WT and mutant when exposed to PEG. Future work will analyze the promoters of candidate genes for the OsSR1 DNA-binding motif (CG-1) to identify direct targets of OsSR1. Rice is the model organism for monocots and provides 15% of the calories consumed by humans. This study and other studies based on this work will help in elucidating the functions of this stress-responsive transcription factor, OsSR1, in this important crop plant.
Open Access
Initiation and regulation of iron economy in Arabidopsis thaliana chloroplasts
(Colorado State University. Libraries, 2020) Kroh, Gretchen Elizabeth, author; Pilon, Marinus, advisor; Reddy, Anireddy, committee member; Bush, Daniel, committee member; Bedinger, Patricia, committee member; Argueso, Cristiana, committee member
Iron (Fe) is biologically important for all organisms because of its role as a protein cofactor which provides redox and catalytic functions. Fe cofactors come in 3 different forms (Fe-S clusters, heme, and non-heme Fe). Plants have a stronger requirement for Fe than non-photosynthetic organisms because the chloroplast has a high demand for Fe. Plants are commonly Fe deficient because soil Fe is typically found in the non-bioavailable, ferric (Fe3+) form, which limits plant growth in natural and agricultural settings. When grown on soils where Fe availability is low, plants can increase Fe uptake and use Fe more efficiently. The leaf response to Fe limitation in the model plant, Arabidopsis thaliana, is the topic of my dissertation. As a major contribution to a larger study, I first characterized the transcriptional response for specific leaf genes to Fe deficiency in the leaf and found that transcripts for abundant chloroplast Fe proteins were down-regulated, suggesting an Fe economy response. Specifically, photosynthetic electron transport and chloroplast Fe-S assembly were targeted for down-regulation. Fe deficiency affects photosynthesis and chloroplast Fe protein expression. I characterized a photosynthesis mutant and found that the regulation of Fe protein expression is maintained, suggesting that loss of electron transport does not trigger down-regulation of Fe protein expression. By using RNA-seq, I analyzed genome-wide transcriptomic changes to identify co-regulated transcripts early in the Fe economy response, including candidate transcription factors. The transcriptional responses in wild type Fe limited plants and a chloroplast Fe-S assembly mutant were independent of each other, suggesting that Fe-S assembly does not generate a signal to regulate chloroplast Fe proteins. The novel insights provided in this dissertation form a foundation for understanding how photosynthetic organisms cope with Fe limitation. From an applied perspective, the results of this dissertation open new avenues to minimize effects of Fe deficiency in agricultural settings.
Open Access
Investigating the biochemistry and genetics of chrysolaminarin metabolism in a model marine diatom
(Colorado State University. Libraries, 2017) Caballero, Michael Adan, author; Peers, Graham, advisor; Belisle, John, committee member; Bush, Daniel, committee member; Prasad, Ashok, committee member
To view the abstract, please see the full text of the document.
Open Access
Investigation of multiple interspecific reproductive barriers in Solanum section Lycopersicon
(Colorado State University. Libraries, 2016) Baek, You Soon, author; Bedinger, Patricia A., advisor; Bush, Daniel, committee member; Argueso, Cris, committee member; Brick, Mark, committee member
Interspecific reproductive barriers (IRBs) act to prevent hybridization between close relatives and provide insight on how species maintain their integrity in nature. Wild tomato species (Solanum Section. Lycopersicon) are useful for studying IRBs. The monophyletic tomato clade includes 13 closely related species that possess a variety of mating systems and complex IRBs. IRBs can be classified according their operation during reproduction in plants; IRBs occurring before mating (premating prezygotic barriers), those operating after mating but before fertilization (postmating prezygotic barriers), and those acting after fertilization (postzygotic barriers). In the tomato clade, postmating prezygotic barriers regulating pollen tube growth in pistils are known to be important for preventing hybridization. Interspecific pollen rejection frequently displays the SI x SC rule, in which crosses between self-incompatible (SI) species and self-compatible species (SC) are successful in one direction but the reciprocal crosses fail, resulting in unilateral incompatibility (UI). This implies that mechanisms involved in SI and IRB systems overlap. I tested multiple aspects of IRBs in the tomato clade at different points in reproduction. First, I assessed pollen grain size and style length among nine species in the tomato clade to test the hypothesis that larger pollen is required to traverse longer styles. I found no correlation between pollen grain size and style length, and thus it is unlikely that either of these factors act as a reproductive isolating mechanism among the wild tomato species. Second, I assessed pollen-pistil interactions in interspecific crosses among 13 species of tomato species in order to test the SI x SC rule in the tomato clade (Solanum sect. Lycopersicon). I found that the SI x SC rule was generally followed at the species level, but exceptions to the SI x SC rule were observed with more recently evolved SC populations. My results further revealed differences in strength of both pistil and pollen IRBs in the tomato clade. Third, I assessed a series of IRBs between geographically co-occurring species of the tomato clade from 12 sympatric sites. My previous study assessed the relationship between interspecific populations that do not share range overlap, so this study was performed to understand IRBs in an ecologically relevant context. I did not find consistent reductions in stigma exsertion (which would contribute to lower outcrossing rates) of the SC species Solanum pimpinellifolium from sympatric sites, suggesting that this floral trait is unlikely to act as a reproductive barrier in this species. In six instances, I detected strong post-mating prezygotic IRBs, in which pollen tubes of SC S. pimpinellifolium were consistently rejected by pistils of their SI sympatric partner. I also identified a possible case of conspecific pollen preference (relatively slower interspecific pollen tube growth) in one sympatric species pair. In cases where prezygotic IRBs were not observed, I mostly found strong post-zygotic IRBs in the form of abnormal seed development in which embryos only progressed to the globular stage. Although I identified multiple IRBs between sympatric pairs, normal seed was formed in three crosses resulting in F1 hybrid plants. These studies suggest that most sympatric populations in the tomato clade exhibit a combination of prezygotic and postzygotic IRBs that prevent hybridization between species, although there may be exceptions. Finally, I investigated whether a low activity S-RNase protein (SI pistil factor) is involved in IRBs in the wild SC species Solanum neorickii. Populations of S. neorickii located at northern and southern margins of the distribution reject interspecific pollen and express a low activity S-RNase protein, whereas those in the center of the species range do not reject interspecific pollen and lack expression of the S-RNase. To determine whether this low activity S-RNase is sufficient for the observed IRB (or if another factor is involved), I crossed individuals from populations which show difference in S-RNase expression and interspecific pollen tube rejection and generated F1 hybrids and F2 lines. In the F2, I observed individuals that express S-RNase and reject interspecific pollen tubes, and those that lack S-RNase and are not capable of rejecting interspecific pollen tubes, as expected. However, I also observed individuals that express S-RNase but do not reject interspecific pollen tubes. These findings suggest that a low activity S-RNase is necessary but not sufficient to reject interspecific pollen tubes in S. neorickii. The findings presented in my dissertation research are major advances that aid in our understanding of reproductive barriers in wild populations. Further, studies of reproductive barriers in tomato, a major food crop, have important implications for agronomic improvement. Many QTL conferring disease resistance, fruit quality and other important traits have been introgressed into cultivated tomato from wild species, but the success of introgression is often inhibited by reproductive barriers.