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
| dc.contributor.author | Guignardi, Zackary S., author | |
| dc.contributor.author | Pilon-Smits, Elizabeth, advisor | |
| dc.contributor.author | Pilon, Marinus, committee member | |
| dc.contributor.author | Santangelo, Thomas, committee member | |
| dc.date.accessioned | 2017-09-14T16:05:52Z | |
| dc.date.available | 2017-09-14T16:05:52Z | |
| dc.date.issued | 2017 | |
| dc.description.abstract | 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. | |
| dc.format.medium | born digital | |
| dc.format.medium | masters theses | |
| dc.identifier | Guignardi_colostate_0053N_14361.pdf | |
| dc.identifier.uri | https://hdl.handle.net/10217/183990 | |
| dc.language | English | |
| dc.language.iso | eng | |
| dc.publisher | Colorado State University. Libraries | |
| dc.relation.ispartof | 2000-2019 | |
| dc.rights | Copyright and other restrictions may apply. User is responsible for compliance with all applicable laws. For information about copyright law, please see https://libguides.colostate.edu/copyright. | |
| dc.subject | plant | |
| dc.subject | sulfate | |
| dc.subject | yeast | |
| dc.subject | selenium | |
| dc.subject | hyperaccumulator | |
| dc.subject | tissue culture | |
| dc.title | 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 | |
| dc.type | Text | |
| dcterms.rights.dpla | This Item is protected by copyright and/or related rights (https://rightsstatements.org/vocab/InC/1.0/). You are free to use this Item in any way that is permitted by the copyright and related rights legislation that applies to your use. For other uses you need to obtain permission from the rights-holder(s). | |
| thesis.degree.discipline | Biology | |
| thesis.degree.grantor | Colorado State University | |
| thesis.degree.level | Masters | |
| thesis.degree.name | Master of Science (M.S.) |
Files
Original bundle
1 - 1 of 1
Loading...
- Name:
- Guignardi_colostate_0053N_14361.pdf
- Size:
- 2.13 MB
- Format:
- Adobe Portable Document Format