Browsing by Author "Pilon-Smits, Elizabeth, advisor"

Now showing 1 - 13 of 13

Open Access
Ecological aspects of plant selenium hyperaccumulation: effects of selenium hyperaccumulation on plant-plant interactions
(Colorado State University. Libraries, 2016) Mehdawi, Ali Farag El, author; Pilon-Smits, Elizabeth, advisor; Pilon, Marinus, committee member; Paschke, Mark, committee member; Vivanco, Jorge, committee member
Hyperaccumulators are plants that accumulate toxic elements to extraordinary levels. Selenium (Se) hyperaccumulators such as Astragalus bisulcatus and Stanleya pinnata can contain 0.1-1.5% of their dry weight in Se (1,000 - 15,000 mg Se kg-1 DW), levels toxic to most other organisms. Selenium promotes hyperaccumulator growth and also offers the plant several ecological advantages through negative effects on Se-sensitive partners. Previous work has shown that high tissue Se levels reduce herbivory and pathogen infection. On the other hand, hyperaccumulators may offer an exclusive niche for Se-tolerant ecological partners. The focus of this dissertation study was on the effects of Se hyperaccumulation on plant-plant interactions. The first Chapter presents a literature review of the phenomenon of Se hyperaccumulation, how Se hyperaccumulators are different from other plants, and an overview of previous studies on the effects of hyperaccumulated Se on ecological processes related to herbivore-plant interactions, microbe-plant interactions and pollinator-plant interactions. In addition, evolutionary aspects of Se hyperaccumulation are discussed, and their implications for their ecological partners. The findings presented in this overview formed the platform for the experiments carried out in this dissertation research, on the topic of plant-plant interactions. In Chapter 2, experiments are described to address the question whether Se hyperaccumulation can negatively affect neighboring plants. Soil collected around hyperaccumulators on a seleniferous field site was measured and shown to contain more Se (up to 266 mg Se kg-1) than soil around non-hyperaccumulators. Vegetative ground cover was somewhat lower around Se hyperaccumulators compared to non-hyperaccumulators. Thus, Se hyperaccumulators may increase surrounding soil Se concentration (phytoenrichment). The enhanced soil Se levels around hyperaccumulators were shown to impair growth of a Se-sensitive plant species, Arabidopsis thaliana, pointing to a possible role of Se hyperaccumulation in elemental allelopathy. In Chapter 3, potential positive effects of hyperaccumulator Se on neighboring plants are explored. It was found for two plant species, Artemisia ludoviciana and Symphyotrichum ericoides, that growing next to Se hyperaccumulators increased their Se content 10-20 fold (up to 800-2,000 mg Se kg-1 DW) compared to when they were growing next to non-accumulators. Moreover, these neighbors of hyperaccumulators were 2-fold bigger, showed 2-fold less herbivory damage and harbored 3-4 fold fewer arthropods than when growing next to non-hyperaccumulators. When used in laboratory choice and non-choice grasshopper herbivory experiments, Se-rich neighbors of hyperaccumulators experienced less herbivory and caused higher grasshopper Se accumulation (10-fold) and mortality (4-fold). These results suggest that Se hyperaccumulators can facilitate the growth of Se-tolerant neighboring plants. The fourth Chapter describes a more controlled greenhouse pot cocultivation study that investigated how Se affects relationships between Se hyperaccumulators (A. bisulcatus and S. pinnata) and related non-accumulator species (A. drummondii and S. elata), in terms of how these plants influence their neighbor’s Se accumulation and growth. Selenium affected growth differently in hyperaccumulators and nonaccumulators: The hyperaccumulators performed 2.5-fold better on seleniferous than non-seleniferous soil, and grew up to 4-fold better with increasing Se supply, while the non-accumulators showed opposite results. Both hyperaccumulators and non-accumulators could affect growth (up to 3-fold) and Se accumulation (up to 6-fold) of neighboring plants. The mechanisms for these effects are largely unknown but may involve concentration of soil Se via exudation, root turnover and litter deposition. Exudate of selenate-supplied A. bisulcatus was shown by x-ray absorption spectroscopy to contain mainly C-Se-C. In conclusion, Se hyperaccumulators may enhance the soil Se levels under their canopy, and also convert inorganic Se to organic Se. The Se-enriched soil around hyperaccumulators enhances Se levels in neighboring plants, which may negatively affect Se-sensitive neighboring plants via toxicity, but facilitate Se-tolerant neighbors through reduced herbivory. The latter is an interesting finding, as it constitutes facilitation via enrichment with a non-essential element. It is also interesting that Se enrichment of neighbors by hyperaccumulators can result in competition when neighbors are Se-sensitive and in facilitation when neighbors are Se-tolerant. Via these competitive and facilitating effects, Se hyperaccumulators may affect plant species composition and, consequently, higher trophic levels. Hyperaccumulators may favor Se resistant species at different trophic levels, while selecting against Se sensitive species. If indeed Se hyperaccumulators affect soil Se distribution and speciation and local species composition and Se tolerance, Se hyperaccumulators may play an important role in Se entry into and Se cycling through their seleniferous ecosystems.
Open Access
Ecological effects of selenium hyperaccumulation on plant community structure and potential implications for selenium cycling
(Colorado State University. Libraries, 2019) Reynolds, Ray Jason Bixler, author; Pilon-Smits, Elizabeth, advisor; Paschke, Mark, committee member; von Fischer, Joseph, committee member; Steingraeber, David, committee member
To view the abstract, please see the full text of the document.
Open Access
Ecological interactions involving plant selenium hyperaccumulation
(Colorado State University. Libraries, 2010) Quinn, Colin Francis, author; Pilon-Smits, Elizabeth, advisor; Paschke, Mark, committee member; Seshadri, Arathi, committee member; Steingraeber, David, committee member
To view the abstract, please see the full text of the document.
Open Access
Interplay between selenium hyperaccumulator plants and their microbiome
(Colorado State University. Libraries, 2016) Cochran, Alyssa T., author; Pilon-Smits, Elizabeth, advisor; Leach, Jan, committee member; von Fischer, Joseph, committee member; Stromberger, Mary, committee member
The plant microbiome includes all microorganisms that occur on the plant root (rhizosphere) and shoot (phyllosphere) or inside plants (endosphere). Many of these microbes benefit their host by promoting growth, helping acquire nutrients or by alleviating biotic or abiotic stress. In addition to its intellectual merit, better knowledge of plant-microbiome interactions is important for agriculture and medicine. Microbiome studies are gaining popularity in multiple research areas, particularly due to advances in next generation sequencing, which has advantages over cultivable methods by revealing the complete microbial community. Still relatively little is known about the microbiomes of plants with extreme properties, including plants that hyperaccumulate (HA) toxic elements such as selenium (Se). Selenium HAs may contain up to 1.5% of their dry weight in Se, which can cause toxicity to herbivores and pathogens as well as neighboring plants. Many advances are yet to be made with regard to the interaction of Se and the plant microbiome: does plant Se affect microbial diversity and composition, and do plant-associated microbes affect plant Se accumulation? The first chapter of this thesis will discuss aspects of the plant microbiome as well as the discoveries to date with regard to plant-associated microbes and Se, mostly explored through culture-dependent methods. Selenium HA appear to harbor equally diverse endophytic microbial communities as non-hyperaccumulators. Thus, plant Se does not impair associations with microbes. A variety of microbes have been isolated from plants or soil in seleniferous areas, including some bacteria and fungi with extreme Se tolerance. Inoculation of plants with individual strains or consortia of microbes was able to promote plant growth, Se uptake and/or Se volatilization. Thus, microbes may facilitate their host’s fitness in seleniferous areas. Exploiting and optimizing plant-microbe associations may facilitate applications like phytoremediation (bio-based environmental cleanup) or biofortification (nutritionally fortified crops). Plant-derived microbial isolates may also be applicable without their plant host, e.g. for cleanup of wastewaters. Culture-dependent studies have dominated the plant-microbe interactions research in regards to hyperaccumulators thus far, painting an elaborate but incomplete picture. In the second chapter of this thesis, we use a mix of culture based and culture-independent methods to investigate the bacterial rhizobiome of selenium Se HAs. Using 16S rRNA Illumina sequencing, we show that the rhizobiomes of Se HAs are significantly different from non-accumulators from the same naturally seleniferous site, with a higher occurrence of Pedobacter and Deviosa surrounding HAs. In addition, we found that HAs harbor a higher species richness when compared to non-accumulators on the same seleniferous site. Thus, hyperaccumulation does not appear to negatively affect rhizobiome diversity, and may select for certain bacterial taxa in the rhizobiome. The bacterial isolates, independent from site or host plant species were in general extremely resistant to toxic concentrations of Se (up to 200mM selenate or selenite) and could reduce selenite to elemental Se. Thus, microbial Se resistance may be widespread and not be under selection by Se HAs. In future studies it will be interesting to further investigate the mechanisms by which Se HA species similarly shape their rhizobiome; this is perhaps due to Se-related root exudates. Future studies may also focus on elucidating the effects of microbes on plant Se accumulation and tolerance.
Open Access
Phytoremediation and biofortification potential of Cannabis sativa L.
(Colorado State University. Libraries, 2019) Stonehouse, Gavin C., author; Pilon-Smits, Elizabeth, advisor; Pilon, Marinus, committee member; Ippolito, Jim, committee member
Selenium (Se) is a micronutrient, but toxic at high levels. Both Se deficiency and toxicity are problems worldwide. I studied the potential of hemp (Cannabis sativa L.) for Se environmental cleanup (phytoremediation) and for accumulating elevated levels of this healthy micronutrient (Se biofortification). Hemp properties attractive for phytoremediation are fast growth, high biomass, hardiness and economic value. Furthermore, hemp produces highly nutritious seeds, of interest for Se biofortification. The first Chapter of this thesis reviews Cannabis sativa's history, biological attributes and applications, as well as the technologies of phytoremediation and biofortification, and plant Se metabolism. The second Chapter presents experimental data on two hemp studies. The first was a field survey of Se accumulation in hemp grown across Colorado, and in commercial hemp products. The second study involved controlled greenhouse experiments to study hemp Se tolerance, accumulation and metabolism. Hemp field surveys in four naturally seleniferous (Se-rich) agricultural areas in Colorado, U.S.A. found 15-25 µg Se/g in seed (intact or dehulled) and 5-10 µg Se/g dry weight in flowers and leaves. Hemp beer contained 42 µg Se/L. Considering the U. S. recommended daily allowance (RDA) of 55-75 ug Se, one bottle of hemp beer provides 25%, and 4 gram hemp seed (a half tablespoon) provides 100% of the RDA. In controlled greenhouse experiments, hemp was further characterized for Se tolerance, accumulation and Se speciation. Effects of Se on photosynthesis and cannabinoid and terpenoid levels were also analyzed. At the seedling level, hemp showed high selenate tolerance (up to 160 µM) and accumulation (up to 1,400 mg Se/kg shoot dry weight). Mature hemp was completely tolerant up to 40 µM selenate and accumulated up to 200 mg Se/kg DW in leaves, flowers and seeds. Seeds were found to contain free (water-extractable) selenomethionine and methyl-selenocysteine, superior forms for Se biofortification, reported to have anticarcinogenic properties for consumers. Hemp production of medicinal cannabidiol (CBD) and terpenoids was not affected by Se. Selenium enhanced potassium levels in seeds, and thus their nutritional value; other nutrient levels were unaffected. It can be concluded from these studies that hemp shows promise for Se phytoremediation and can produce Se-biofortified dietary products; Se does not affect levels of valuable secondary plant compounds, nor does it negatively affect nutritional quality of seeds. These findings are of significance in view of the widespread and rapidly expanding cultivation of hemp in seleniferous areas across the U.S.A. and Canada.
Open Access
Phytoremediation with hemp (Cannabis sativa L.): a look at hemp's potential for environmental cleanup and economic recovery
(Colorado State University. Libraries, 2022) Abernathy, Susan M., author; Pilon-Smits, Elizabeth, advisor; Pilon, Marinus, committee member; Qian, Yaling, committee member
The aim of this thesis study was to test hemp's (Cannabis sativa L.) potential for phytoremediation (environmental clean-up). I tested hemp for tolerance and accumulation of four inorganic pollutants, to evaluate its remediating performance. Hemp has many properties that would make it a likely candidate for phytoremediation however, due to recent regulations, research of this versatile plant has been limited. Phytoremediation is a process of cleaning polluted sites using plants. In this clean-up method, plants may stabilize the pollutant in situ, or take-up the pollutant into the plant tissue. In the latter, there are a few different fates for the pollutant that include degradation, metabolization, sequestration, and/or volatilization. Phytoremediation is a clean process that reestablishes an onsite ecosystem and is a competitive alternative to more conventional scrape-and-remove methods. Hemp is a hardy, fast growing species that produces high biomass. Hemp has deep roots that can be used to reach pollutants deep in the ground. These properties make hemp a potential choice for phytoremediation. Contaminated sites create harsh growing conditions that require hardy plant properties in order for a species to survive. An added benefit to using hemp for remediation is the many economic uses of hemp biomass. Each part of the hemp plant can be used to make goods such as clothing, building material, cosmetics, lotions, animal bedding, fragrances, and medicinal products that have therapeutic qualities. In addition, hemp seeds are nutritious and can be added to the diet. In chapter one of this thesis, phytoremediation is reviewed to explain the remediation process. This review includes explaining the different phytotechnologies that are employed by plants which depend on the plant used and the type of pollutant encountered. Chapter one also reviews hemp, its history, biology, and the properties that make it a viable choice for phytoremediation. Chapter two of this thesis is an experimental chapter presenting data for testing hemp seedlings with four different oxyanions: arsenate (As), molybdate (Mo), vanadate (V), and tungstate (W). The parameters considered were biomass, chlorophyll content, chlorophyll fluorescence, pollutant accumulation levels, and pollutant fate. Brassica juncea (Indian mustard) was used as a reference phytoremediation species. The findings of this thesis study present promising results for hemp as a potential remediator. Arsenic was found to accumulate in the root at levels up to 2700 mg kg-1 DW. Tungsten also accumulated in the root at levels up to 3100 mg kg-1 DW. In both tests, hemp performed well, judged from photosynthetic measurements and relative chlorophyll content, but reduced biomass started at treatments with 3 and 24 mg As L-1 in the shoot and root respectively, and 40 and 80 mg W L-1 in the shoot and root, respectively. Molybdenum accumulated in the shoot at levels up to 4900 mg kg-1 DW and in the root at levels up to 2600 mg kg-1 DW. Biomass reduction of Mo started at treatment with 40 mg Mo L-1 for both shoot and root, while photosynthetic measurements and relative chlorophyll content remained unchanged. Lastly, V accumulated in the root at levels up to 2100 mg V kg-1 DW. Interestingly, hormesis (stimulated growth) was observed in hemp supplied with V: biomass increased at all tested levels. From this study, it was concluded that hemp may have potential for phytoremediation in cleaning contaminated sites with the four elements tested. Hemp performed competitively with the popular phytoremediation species, Indian mustard (Brassica juncea L.) in all levels tested for Mo, V, and W. Hemp's economic recovery with clean post-harvest biomass may offset phytoremediation costs giving this species a unique advantage over other popular phytoremediation choices.
Open Access
Plant-microbe interactions of selenium hyperaccumulators: effects on plant growth and selenium metabolism
(Colorado State University. Libraries, 2012) Lindblom, Stormy Dawn, author; Pilon-Smits, Elizabeth, advisor; Leach, Jan, committee member; Wangeline, Ami, committee member; von Fischer, Joe, committee member
To view the abstract, please see the full text of the document.
Open Access
Selenium accumulation in plants and implications for human health: a survey of molecular, biochemical, and ecological cues
(Colorado State University. Libraries, 2022) Lima, Leonardo Warzea, author; Pilon-Smits, Elizabeth, advisor; Schiavon, Michela, committee member; Pilon, Marinus, committee member; Antunes, Mauricio, committee member; Paschke, Mark, committee member
To view the abstract, please see the full text of the document.
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 member
The 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.
Open Access
Selenium uptake, differentiation and metabolism in hyperaccumulator Stanleya pinnata
(Colorado State University. Libraries, 2013) Harris, Jonathan, author; Pilon-Smits, Elizabeth, advisor; Pilon, Marinus, committee member; Peers, Graham, committee member; Ward, Sarah, committee member
Selenium (Se) is a biologically essential element for many animals, some prokaryotes and algae. However, even in organisms that require Se, the range between sufficiency and toxicity for Se is narrow. Although there are no reports of a Se requirement or selenoproteins in higher plants, there are species that appear endemic to seleniferous soil and concentrate Se in their leaves to levels exceeding 1000 mg kg-1 dry weight. These plants are known as Se hyperaccumulators and have an exceptional ability to tolerate and enrich themselves with this toxic element. As a result of the Se concentrations in their tissues, Se hyperaccumulators are extremely toxic to most organisms. Studies have found that Se hyperaccumulation protects these plants from many herbivores and pathogens as an "elemental defense." Some of these hyperaccumulators have been studied for their use in phytoremediation of naturally occurring and anthropogenically contaminated seleniferous soils. Although the slow growth of most hyperaccumulators limits their direct application for phytoremediation, they can be utilized as a source of genes to genetically enhance Se accumulation and tolerance in popular phytoremediator species. The goal of this study is to better characterize the uptake, metabolic fate and molecular mechanisms responsible for Se tolerance in Stanleya pinnata, a hyperaccumulator in the Brassicacae. Two main techniques were utilized: physiological experiments followed by elemental analysis to characterize Se uptake and interactions with the related element sulfur (S), and Illumina sequencing of the transcriptomes of Stanleya pinnata and related non-hyperaccumulator Stanleya elata. The first chapter presents a literature review of Se hyperaccumulation: what is known about Se assimilation in higher plants, and some unique characteristics of hyperaccumulators. The metabolism of Se through the sulfate assimilation pathway is described, and known mechanisms of Se tolerance and accumulation in representative plants are reviewed. In addition, some of the previous work on Stanleya is reviewed, including a number of studies that have shown ecological benefits of Se hyperaccumulation. Known beneficial genes for Se tolerance and accumulation are discussed in the context of phytoremediation. In chapter 2, Se-specific uptake was tested in two ecotypes of S. pinnata, and contrasted with related non-hyperaccumulator Brassica juncea. To test for Se specificity of sulfate transporters, plants were supplied with varying concentrations of selenate and two concentrations of sulfate. The results showed that S. pinnata is able to take up large amounts of Se, even at exceedingly low supplied Se:S ratios. In addition, S. pinnata preferentially mobilized large amounts of Se to young leaves, without commensurate mobilization of S. These trends were not observed in the non-hyperaccumulator B. juncea, which showed dramatically reduced Se uptake under elevated sulfate supply. Moreover, there was no evidence of preferential allocation of Se to young tissues in B. juncea. Taken together, these findings support the hypothesis that Stanleya contains transporters with an increased specificity for Se, allowing it to take up preferentially and mobilize Se over S. Since previous work has shown that molybdate may be taken up in part by plant sulfate transporters, this element was also monitored. It was observed that increasing supply of selenate and sulfate significantly reduced the molybdenum (Mo) content of leaves in S. pinnata. In contrast, B. juncea showed an increase in Mo content with increases in supplied selenate. In the experiment described in Chapter 3, Illumina sequencing was performed to compare the root and shoot transcriptomes of hyperaccumulator S. pinnata and non-hyperaccumulator S. elata in the presence or absence of selenate. An overview is presented of the overall transcriptome response patterns, followed by a more detailed analysis of transcripts involved in S/Se metabolism. In the presence of Se, 40 of the 56 S/Se-related genes were more highly expressed in S. pinnata than S. elata. Particularly promising findings include a vastly upregulated root sulfate/selenate transporter (Sultr1;2) and ATP sulfurylase (APS2). Lastly, some preliminary findings are presented from several biochemical approaches used to further investigate S. pinnata hyperaccumulation mechanisms. Organic forms of Se were investigated in S. pinnata and S. elata using a newly developed liquid chromatography mass spectrometry (LC-MS) method. It was shown that S. pinnata accumulates significant amounts of selenocystathionine as well as methyl-selenocysteine. Moreover, activities of selenocysteine lyase (SL) and cysteine desulfurase (CysD) were investigated in S. pinnata and S. elata, which revealed strong SL activity in the hyperaccumulator. The possible role of this enzyme in Se hyperaccumulation remains to be elucidated. Finally, superoxide dismutase activities were compared between the two species in relation to Se supply. Stanleya pinnata and other Se hyperaccumulators may be valuable resources for genes involved in Se tolerance and hyperaccumulation, to create genetically engineered plants for phytoremediation purposes. In addition to the potential environmental benefits, understanding potential biological roles for Se and its metabolism in these plants may have broad applications for human health. Many organic seleno-compounds have been studied for their anti-carcinogenic properties in multiple systems and types of cancer. Efficacy of these Se compounds appears to vary based on the form of Se. Plants capable of creating different forms of organic Se may become a valuable pharmaceutical resource.
Open Access
Studies on selenium hyperaccumulator Stanleya pinnata and nonaccumulator Stanleya elata (Brassicaceae): functional characterization of selenate transporter SULTR1;2 in yeast and development of a micropropagation protocol
(Colorado State University. Libraries, 2017) Guignardi, Zackary S., author; Pilon-Smits, Elizabeth, advisor; Pilon, Marinus, committee member; Santangelo, Thomas, committee member
Stanleya pinnata is an herbaceous perennial species in the family Brassicaceae native to the western United States. This species is classified as a selenium (Se) hyperaccumulator, and can be found thriving on Se-rich soils. Selenium hyperaccumulators are plant species that have the capacity to accumulate Se over 1,000 mg kg-1 dry weight in their tissues, concentrations toxic to non-accumulator plant species as well as to herbivores and pathogens, which may explain why plants hyperaccumulate Se. Due to the chemical similarity of Se to sulfur (S), Se is believed to be transported and metabolized by the same proteins and enzymes, including sulfate transporters and the sulfate assimilation pathway. Selenate (SeO42-), the predominant available form of Se in soil, is transported into the roots mainly via the high-affinity membrane transporter SULTR1;2. While most plants do not appear to discriminate between selenate and sulfate, and the two compounds compete for uptake, selenate uptake in Se hyperaccumulators is less inhibited by high sulfate concentrations. Since SULTR1;2 is the main portal of entry for selenate into the plant, it may be hypothesized that SULTR1;2 from the Se hyperaccumulator S. pinnata has intrinsic properties that allow this species to discriminate between sulfate and selenate and preferentially take up selenate. One of the objectives of this thesis project was to test this hypothesis, by means of functional characterization of SULTR1;2 from S. pinnata and from control species Stanleya elata, and Arabidopsis thaliana in the YSD1 yeast mutant which lacks its native sulfate transporters. A secondary objective in this thesis project was to develop a micropropagation protocol for Stanleya. In order to effectively study Se hyperaccumulation in a laboratory setting, sufficient numbers of S. pinnata and S. elata plants need to be available. However, due to low rates of seed germination, vernalization requirements, self-incompatibility, and ineffectiveness of propagation by cuttings, conventional propagation methods via seed or vegetative cuttings severely limit the number of plants that can be cultivated at a time. In order to overcome these limits, a tissue culture micropropagation protocol for leaf explants of S. pinnata and S. elata was developed. This protocol will allow for the rapid reproduction of both Stanleya species, not only to be used in laboratory experiments, but also in industrial applications such as Se phytoremediation projects, as well as for horticultural and native landscaping purposes. The first chapter of this thesis reviews plant Se uptake and metabolism, offering an overview of the current understanding of the Se assimilation pathway in plants, including mechanisms of accumulation and tolerance unique to Se hyperaccumulators. This chapter also outlines key proteins and enzymes in the Se assimilation pathway that are candidates for future experiments to determine the mechanisms of Se hyperaccumulation. The second chapter describes the results from yeast studies, characterizing the selenate and sulfate transport capabilities of SULTR1;2 from hyperaccumulator S. pinnata and non-accumulators S. elata, and A. thaliana, and their selenate specificity, as judged from the effects of sulfate competition on selenate uptake. Interestingly, yeast transformed with SULTR1;2 from S. pinnata (SpSultr1;2) showed less inhibition of selenate uptake by high sulfate concentration, indicating that this species' selenate selectivity may be facilitated by the SULTR1;2 protein. While apparently more Se-specific, yeast transformed with SpSultr1;2 overall took up less Se when compared to yeast expression SULTR1;2 from non-accumulators. It is feasible that a mutation that changes the substrate specificity of SpSULTR1;2 also reduced its overall activity. In S. pinnata, SpSultr1;2 transcript was found in earlier studies to be ~10-fold up-regulated when compared to S. elata, which may compensate for decreased activity. Identification of a selenate-specific transporter has applications for Se phytoremediation and biofortification. Constitutive overexpression of a hyperaccumulator selenate transporter in other plant species may increase their uptake of Se, even in the presence of high environmental S levels. The third chapter of this thesis outlines the development of a fast and efficient tissue culture micropropagation protocol for S. pinnata and S. elata. Through the testing of multiple concentrations of hormones on in vitro callus formation, shoot induction and elongation, and root formation, followed by ex vitro acclimatization, both species of Stanleya were shown to be very amenable to micropropagation. Both exhibited rapid callus, shoot, and root induction under a wide range of 1-napthaleneacetic acid (NAA), 6-benzylaminopurine (BAP), and indole-3-butyric acid (IBA) concentrations. Future experiments could explore the genetic transformation of S. elata plants with genes from S. pinnata to test their importance for Se accumulation and tolerance in this related non-accumulating species. This micropropagation protocol also opens up the possibility to cultivate the Stanleya species at a large scale for multiple applications including biofortification, phytoremediation, and native landscaping.
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, advisor
NifS-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.
Open Access
Transcriptome and elemental analysis of the selenium hyperaccumulator Stanleya pinnata and non-accumulator Stanleya elata
(Colorado State University. Libraries, 2015) Wang, Jiameng, author; Pilon-Smits, Elizabeth, advisor; Sloan, Daniel, committee member; Stargell, Laurie, committee member
To view the abstract, please see the full text of the document.