Browsing by Author "Pilon, Marinus, advisor"
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Item 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 memberCopper (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.Item Open Access Analyzing genetic response mechanisms associated with copper homeostasis in Populus trichocarpa using a bioinformatics approach(Colorado State University. Libraries, 2013) Patterson, Eric, author; Pilon, Marinus, advisor; Bedinger, Patricia, committee member; Jahn, Courtney, committee member; Walters, Christina, committee memberCopper is an essential micronutrient for plants and plays an important role in photosynthesis, respiration, hormone signaling, cell wall structure and wound healing. Copper deficiency can cause chlorosis, leaf curling, and weakened stems. It is proposed that under copper deficient conditions plants down regulate genes whose proteins use copper as a cofactor but also play an "unessential" role for the plants survival, thereby preserving copper for more "essential" proteins like plastocyanin or cytochrome-C oxidase. Down-regulation of "unessential" genes is performed by the copper microRNAs miR307, miR398, and miR408. This thesis increases our understanding of copper homeostasis in plants by analyzing the transcriptomic response of Populus trichocarpa to copper deficiency in four vegetative organs and applies this knowledge to the study of multi-copper oxidases. Organs have drastically different responses to copper deficiency with few genes being systemically differentially expressed and most genes that are differential expressed only are in one organ. Our data also show that not all genes are regulated to the same extent. Genes that are already highly expressed (>50 RPKM) under copper-sufficient conditions are only up-regulated 1- to 4-fold, while low expressed genes can be up-regulated as much as 8-fold. We go on to describe 25 unannotated genes as laccases based on their sequence similarity with known laccases from Arabidopsis and Populus. The laccases break up into seven phylogenetically distinct groups. Each of the seven groups have a distinct expression pattern across the four organs in response to copper deficiency that seems to be mediated by Cu-miRNAs miR397 and miR408.Item Open Access Copper transport into the chloroplast and its implications for copper homeostasis in Arabidopsis thaliana(Colorado State University. Libraries, 2012) Tapken, Wiebke, author; Pilon, Marinus, advisor; Chisholm, Stephen, committee member; Pilon-Smits, Elizabeth, committee member; Reddy, Anireddy S. N., committee memberCopper (Cu) is an essential micronutrient for most aerobic organisms including plants. It is present as Cu+ or Cu2+, which makes it an ideal cofactor for enzymes involved in processes such as photosynthesis and respiration. Plant cuproproteins are almost ubiquitously found in every cell compartment. The blue Cu protein plastocyanin (PC) is believed to bind the majority of Cu ions in green tissues and is essential for higher plants. Cu reaches the thylakoid lumen through the activity of two P1B-type ATPases called PAA1/HMA6 and PAA2/HMA8 (P-type ATPase of Arabidopsis/Heavy-metal ATPase), which are located in the inner chloroplast envelope and the thylakoid lumen respectively. Under Cu limiting conditions, plants have been suggested to prioritize cellular Cu to PC to ensure adequate photosynthesis. This process involves the post-transcriptional down-regulation of seemingly less essential cuproproteins through the activity of a single transcription factor called SPL7 (SQUAMOSA promoter binding protein-like7). The first chapter reviews Cu homeostasis in plants. The research presented in the three experimental chapters of this dissertation is aimed to determine the role of the chloroplast in Cu homeostasis of Arabidopsis thaliana. I report a novel SPL7-independent and chloroplast-specific regulation of the thylakoid-localized Cu transporter PAA2/HMA8. The transporter is most abundant in the absence of Cu and is turned over at higher chloroplastic Cu concentrations. PAA2/HMA8 abundance in Cu deficiency is furthermore controlled by the presence of PC, because in a pc mutant PAA2/HMA8 abundance is always low. The regulation of the transporter likely serves as a checkpoint for the Cu requirements of the thylakoid lumen. I identified two components of the stroma-localized Clp protease (Caseinolytic peptidase) which are involved in PAA2/HMA8 turnover. The Cu status of these mutants is not affected, decreasing the likelihood of a secondary affect of Cu on PAA2/HMA8 in these plants. In the last experimental chapter I summarize relevant results that further describe and characterize PAA1 and PAA2. Most notably, Arabidopsis encodes for a splice-form of PAA1. This much smaller fragment is expressed with a chloroplast targeting sequence and could potentially function as a stromal Cu chaperone.Item 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 memberIron (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.Item Open Access Iron economy in Arabidopsis thaliana rosettes(Colorado State University. Libraries, 2014) Hantzis, Laura, author; Pilon, Marinus, advisor; Jahn, Courtney, committee member; Peers, Graham, committee memberIron is important for plant growth and lack of iron negatively affects crop productivity. When we understand more about how plants prioritize their iron use we can use this knowledge to benefit the people of the world. Plant metabolism can be altered to allocate iron in such a way creating larger and/or healthier edible parts. In the first chapter of this thesis I briefly discuss the biological roles of iron and its function in plants with an emphasis on photosynthesis. In the second chapter I investigated the potential prioritization of iron-dependent proteins in Arabidopsis thaliana plants that were grown hydroponically and exposed to one week of iron deprivation followed by one week of iron resupply. Through the one week of iron depletion the treated plants became visually chlorotic and after one week of iron recovery the treated plants appeared to have fully recovered from the chlorosis. At the end of the recovery week treated plants had significantly less biomass than the control plants yet suffered no indirect effects. To investigate if the decrease in biomass was caused by defects in photosynthetic electron transport chlorophyll fluorescence was measured as well as the photooxidation-reduction values of photosystem I (PSI). Indeed photosynthesis was affected and it was found that there was a decrease in electrons flowing to PSI. With the use of immunoblots it was discovered that the cytochrome b6f complex proteins were strongly affected by iron depletion followed by the subunits of PSI. Furthermore, I found that the abundance of sulfur metabolism proteins decreased in reaction to decreased iron nutrition. In the third chapter I discuss the implications of my findings for plant science and society and I give recommendations for follow up work on this project.Item Open Access Regulation of copper homeostasis in plants: a focus on chloroplastic superoxide dismutases and copper delivery mechanisms(Colorado State University. Libraries, 2009) Cohu, Christopher Michael, author; Pilon, Marinus, advisorCopper (Cu) is an essential micronutrient for higher plant growth and is found in proteins that are important in photosynthesis and respiration. As a cofactor, this trace element is associated with many proteins including plastocyanin, Cu/Zn superoxide dismutase (Cu/ZnSOD), and mitochondrial cytochrome- c oxidase. Due to its redox-active role, Cu is essential for plant life, yet Cu is also dangerous as a free cellular ion and even toxic if in excess. Therefore, delivery and sequestration of Cu must be tightly regulated. The research of this dissertation indicates that sensory mechanisms and signaling pathways exist to coordinate Cu transport and target protein expression based on Cu status. For Arabidopsis and crop species, chloroplastic Cu/ZnSOD is down-regulated during limited Cu availability while at the same time FeSOD is up-regulated. During Cu-limited growth, when Cu/ZnSOD is down-regulated, plastocyanin levels do not change. We suggest that this reduction in Cu/ZnSOD allows for preferential Cu delivery to plastocyanin, which is essential for photosynthesis, while also maintaining chloroplast SOD activity. Cu delivery to Cu/ZnSOD is accomplished by the Cu Chaperone for SOD (CCS). When a CCS loss of function mutant was grown on Cu supplemented soil Cu/ZnSOD and FeSOD activity was not detected. Chloroplast did not exhibit an observable phenotype or photosynthetic deficiencies, even after high light stress treatments. Recent studies have shown that Cu/ZnSODs in the cytosol and chloroplast, along with other Cu proteins, are regulated by Cu via microRNA directed cleavage of Cu protein mRNA. It has also been determined that during Cu-limited growth the SPL7 transcription factor plays a central role in activating Cu-microRNAs and possibly Cu transporters. The research of this dissertation indicates that CCS is also regulated by Cu, mediated by microRNA398, which was not previously predicted by bioinformatic algorithms. Furthermore, data is presented to suggest that SPL7 likely regulates the promoter of FeSOD by activating transcription during limited Cu availability.Item Open Access Regulation of copper transport into and within Arabidopsis thaliana chloroplasts: a focus on copper transport proteins(Colorado State University. Libraries, 2007) Gogolin, Kathryn Amy, author; Pilon, Marinus, advisorCopper is an essential micronutrient that is required for the biological processes of photosynthesis and respiration. Nutrients, such as copper, must travel long distances through several organs and across many membranes before they are incorporated into target enzymes. Plastocyanin is a small, copper containing protein that is located within the thylakoid lumen and is vital for photosynthetic activity in higher plants. In addition chloroplasts contain a second target for copper, the superoxide dismutase enzyme CSD2. Although copper is essential it can also be toxic to the cell, therefore there is tight regulation of ion transport. The objective of the research conducted here is to develop a better understanding of copper homeostasis in plant cells. By focusing on the proteins that are involved in the transport of copper new insight can be gained on the delivery pathways of this metal. In this dissertation, I further characterize P-type ATPase of Arabidopsis 1 (PAA1) and P-type ATPase of Arabidopsis 2 (PAA2). An Arabidopsis Copper Chaperone for Cu,Zn Superoxide dimustase (CCS) is identified as a functional homolog of the yeast copper chaperone for Cu,Zn superoxide dimustase (Ccs1/Lys7). I study the effects of altered CCS expression on copper homeostasis in a plant system and I determine that the Heavy Metal Associated 1 transporter functions to transport a metal other than Cu(I) across the chloroplast envelope which affects photosynthetic activity. Finally, I completed a comprehensive analysis of copper transport protein-protein interactions in Arabidopsis studied by the yeast two-hybrid system. With the data gathered here, I propose several new models for copper homeostasis in Arabidopsis. I suggest that there is regulation of Fe Superoxide Dismutase (FeSOD), CCS, and CSD2 in the chloroplast which is controlled by metal cofactor availability, specifically copper. By utilizing the yeast two-hybrid technique, I have identified two new possible delivery pathways for copper. I believe that CCS can deliver copper to Heavy Metal Associated 5 to aid in cell detoxification or possible long distance transport of the ion. Additionally, I propose that copper is transported directly from PAA1 to PAA2 in the chloroplast for delivery to plastocyanin.Item Open Access Selenium transport in plants with a special focus on selenium hyperaccumulators(Colorado State University. Libraries, 2021) Trippe, Richard Croxall, III, author; Pilon-Smits, Elizabeth, advisor; Pilon, Marinus, advisor; Peebles, Christie, committee member; Schiavon, Michela, committee memberThe aims of this thesis are to synthesize current knowledge of selenium (Se) transport and metabolism in plants and improve understanding of Se transport in a class of plant species known as Se hyperaccumulators (HAs). These Se HAs can accumulate Se at up to 1,000 times higher concentrations than normal plants by utilizing specialized systems of Se transport and metabolism. The first chapter of this thesis constitutes a review of the current knowledge about Se transport and metabolism in plants, with a focus on implications for biofortification and phytoremediation. Selenium is a necessary human micronutrient, and around a billion people worldwide may be Se deficient. This can be ameliorated by Se biofortification of staple crops. Selenium is also a potential toxin at higher concentrations, and multiple environmental disasters over the past 50 years have been caused by Se pollution from agricultural and industrial sources. Phytoremediation by plants able to take up large amounts of Se is an important tool to combat pollution issues. Both biofortification and phytoremediation applications require a thorough understanding of how Se is taken up and metabolized by plants. Selenium uptake and translocation in plants is largely accomplished via sulfur (S) transport proteins. Current understanding of these transporters is reviewed here, and transporters that may be manipulated to improve Se uptake are discussed. Plant Se metabolism also largely follows the S metabolic pathway. This pathway is reviewed here, with special focus on genes that may be manipulated to reduce the accumulation of toxic metabolites or enhance the accumulation of nontoxic metabolites. Finally, unique aspects of Se transport and metabolism in Se HAs are reviewed. Selenium hyperaccumulation mechanisms and potential applications of these mechanisms to biofortification and phytoremediation are presented. The second chapter of this thesis covers the results of experimental studies expressing putative Se HA transporter proteins in a yeast (Saccharomyces cerevisiae) model system. Selenium hyperaccumulators are able to take up Se from the soil at concentrations many times higher than other native vegetation. Hyperaccumulators also show evidence of enhanced Se-specificity relative to sulfur. The mechanism for this Se specificity is investigated in this study. We hypothesize that in hyperaccumulators one or more sulfate transport proteins has evolved greater preference for the Se analog, selenate. This study focuses on putative root-to-shoot sulfate/selenate transport proteins SpSULTR2;1 and SpSULTR3;5 from Se hyperaccumulator Stanleya pinnata (Brassicaceae). The coding regions of these genes were amplified via reverse transcription polymerase chain reaction (RT-PCR), cloned in a yeast expression vector and sequenced. SpSULTR2;1 and SpSULTR3;5 were found to be 678 and 638 amino acids in length, respectively. Both proteins showed the characteristic N-terminal cytosolic domain, 12 membrane-spanning domains, and C-terminal cytosolic STAS (Sulphate Transporter and Anti-Sigma factor antagonist) domains. SpSULTR2;1 and SpSULTR3;5 were assayed for selenate specificity by quantifying their relative selenate and sulfate uptake capacities in baker's yeast (Saccharomyces cerevisiae). This assay was complemented with selenate-dependent growth curves in liquid media and a selenate tolerance assay on solid media. Both SpSULTR2;1 and SpSULTR3;5 were expressed by the yeast cells, determined by dot-blot immunoassay. Expression of SpSULTR2;1 effected a slight but non-significant increase in the Se concentration in the yeast relative to the empty vector control in a 1-hour uptake assay when exposed to 1 mM selenate, but not when exposed to 0.1 mM selenate. SpSULTR3;5 was not able to increase the selenate concentration in the yeast relative to the control in the uptake assay. Yeast expressing SpSULTR2;1 or SpSULTR3;5 demonstrated similar selenate tolerance to empty-vector controls. Expression of SpSULTR3;5 or SpSULTR2;1 also did not significantly affect growth relative to the control in liquid media. In conclusion, more studies are needed to determine with certainty whether SpSULTR2;1 or SpSULTR3;5 have selenate-specific transport capability.Item Open Access The role of CpNifS in selenium and sulfur plant metabolism: implications for phytoremediation and photosynthesis(Colorado State University. Libraries, 2008) Van Hoewyk, Doug, author; Pilon, Marinus, advisor; Pilon-Smits, Elizabeth, advisorNifS-like proteins are a conserved group of proteins that can cleave the sulfur-containing amino acid cysteine in alanine and elemental sulfur (S), and selenocysteine alanine and selenium (Se). In yeast and bacteria, NifS-like proteins are essential for survival because they provide the S for iron(Fe)-S clusters, a prosthetic group that is inserted into various FeS proteins that have a role in electron transfer. Furthermore, NifS-proteins are an essential part of Se metabolism in organisms that require this trace element. The goal of this research was to characterize the function of a chloroplastic NifS-like protein in Arabidopsis thaliana, designated AtCpNifS. As described in this dissertation, overexpression of CpNifS increases plant tolerance to selenate and accumulation of Se. Increased levels of CpNifS prevents toxic incorporation of selenocysteine into proteins, and thus enhances Se tolerance. This may benefit phytoremediation-the use of plants to naturally clean polluted soils and groundwater. In an effort to further the field of phytoremediation, a transcriptome experiment was performed in order to identify other genes and pathways that are involved in responding to Se stress. However, as divulged, plants likely do not require Se for essential metabolism, and the true function of CpNifS is more likely in the maturation of FeS clusters. The knockdown of CpNifS proteins in Arabidopsis using an inducible RNAi approach revealed that chloroplast function and structure became impaired, and that levels of all tested FeS proteins decreased. Consequently, the rate of photosynthetic electron transport, which is dependent on FeS proteins, diminished, and plants became chlorotic and eventually died. Therefore, CpNifS is required for FeS proteins, and is essential for proper photosynthesis and plant growth.