SEARCH · Search NASA

Results for “Phytoremediation”

Search indexed NASA NTRS and DOE OSTI research on propulsion, heat transfer, battery materials and energy systems. Follow report and document links to the original sources.

SourceResearch connectionFrom yearThrough year

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 19 records

All sources NASA NTRS DOE OSTI

DOE OSTI2022

Enhancing the Phytoremediation of Heavy Metals by Combining Hyperaccumulator and Heavy Metal-Resistant Plant Growth-Promoting Bacteria

Heavy metals (HMs) have become a major environmental pollutant threatening ecosystems and human health. Although hyperaccumulators provide a viable alternative for the bioremediation of HMs, the potential of phytoremediation is often limited by the small biomass and slow growth rate of hyperaccumulators and HM toxicity to plants. Here, plant growth-promoting bacteria (PGPB)-assisted phytoremediation was used to enhance the phytoremediation of HM-contaminated soils. A PGPB with HM-tolerant (HMT-PGPB), Bacillus sp. PGP15 was isolated from the rhizosphere of a cadmium (Cd) hyperaccumulator, Solanum nigrum . Pot experiments demonstrated that inoculation with strain PGP15 could significantly increase the growth of S. nigrum . More importantly, strain PGP15 markedly improved Cd accumulation in S. nigrum while alleviating Cd-induced stress in S. nigrum . Specifically, PGP15 inoculation significantly decreased the contents of H 2 O 2 , MDA, and O 2 · − in S. nigrum , while the activities (per gram plant fresh weight) of SOD, APX, and CAT were significantly increased in the PGP15-inoculated plants compared with the control sample. These results suggested that the interactions between strain PGP15 and S. nigrum could overcome the limits of phytoremediation alone and highlighted the promising application potential of the PGPB-hyperaccumulator collaborative pattern in the bioremediation of HM-contaminated soils. Furthermore, the PGP15 genome was sequenced and compared with other strains to explore the mechanisms underlying plant growth promotion by HMT-PGPB. The results showed that core genes that define the fundamental metabolic capabilities of strain PGP15 might not be necessary for plant growth promotion. Meanwhile, PGP15-specific genes, including many transposable elements, played a crucial role in the adaptive evolution of HM resistance. Overall, our results improve the understanding of interactions between HMT-PGPB and plants and facilitate the application of HMT-PGPB in the phytoremediation of HM-contaminated soils.

Zhang, Yong↗

DOE OSTI2022

The impact of tritium phytoremediation on plant health as measured by fluorescence

Phytoremediation, using plants for soil, sediment, or water contaminant clean-up, is an established technology dependent on plant health. Tritium (3H), a radioactive isotope of hydrogen that is generally found in the environment as tritiated water (HTO), is a low-level beta emitter with a half-life of 12.32 years. Chlorophyll fluorescence (CF) for monitoring risk assessment of tritium to plant health was conducted at the Tritium Irrigation Facility (TIF) located on the US Department of Energy's Savannah River Site (SRS) near Aiken, SC. Two fluorometers were evaluated in conjunction with phytoremediation at the 25 –acre TIF where tritiated groundwater is being spray–irrigated on a mixed coniferous/deciduous forested watershed as a means of reducing tritium release to a nearby stream that serves as a tributary to the Savannah River. Tritium activity in irrigated water averaged 104 + 42 pCi mL -1 during the 2003 project. Fluorescence parameters measured by the two fluorometers were well correlated with each other (p < 0.0001). Tritium in water respired from oak leaves ranged up to 1845.13 pCi ml -1 and 2138.22 pCi ml -1 in pine needles. Trees in both the test and control sites were approximately 15 years old. In this report we demonstrated that fluorescence parameters provide an effective way to estimate the impact of HTO on plant health in a noninvasive, extremely rapid, and cost-effective manner. In the current study applying fluorometry, plants within the TIF phytoremediation site exposed to the site tritiated water were not significantly impacted by the tritium phytoremediation based on CF parameters as compared to the control, a nascent non-irrigated site.

63 RADIATION, THERMAL, AND OTHER ENVIRON. POLLUTAN↗

DOE OSTI2025

Phytoremediation potential of Nerium oleander and Salix alba for heavy metal removal in rock-amended soils: a natural and cost-effective approach

Enhanced weathering (EW) through the application of ground rock is a competitive carbon removal strategy. Adoption of this technology at a meaningful scale requires a systematic assessment of its long-term feasibility, especially with regard to soil quality from the application of rock amendments that contain varying levels of heavy metal (loid)s (HM) such as Cu, Ni, Cr, Co, and Pb. The potential accumulation of these metal (loid)s could be an unintended consequence of repeated large-scale EW applications, necessitating careful evaluation for use in croplands. This study explores the idea of using phytoremediation as a natural, low-cost means of remediating rock-amended soils. Specifically, we examined the ability of Nerium oleander and Salix alba species to remove HM from rock-amended soils in their tissues (i.e., leaves, stems, and roots). In this study, the relative abundance of HM accumulation in hyperaccumulator plants followed the order: Si > Rb > Cu > Sn > Cr > Cd > Pb > Ni > Mo > Co > As > Sb > Se > Cs. Our results indicate that HM accumulation in soils treated with rock were significantly below permissible limits set by the Environmental Protection Agency (EPA). Further, in reasonable amounts, some of these HM serve as essential micronutrients required by plants. In fact, we found increased growth and higher biomass for both plants under rock application than without. We further found a significant uptake of most HM in rock-amended soils planted with S. alba. Comparatively, uptake of certain HM like Ni, Mo, Cs, Pb and Cu was relatively higher in the roots of N. oleander. In contrast to N. oleander, S. alba accumulated higher levels of HM in its stems and leaves than in its roots. Interestingly, 31–35% weathering of metabasalt (applied rock) was observed across plant types over a 5-month period. Overall, we conclude that S. alba has a greater potential for phytoremediation in rock-amended soils, although both plants may be useful in remediating soils with varying levels and types of contamination.

Ibrahim, Naira [Jackson State Univ., Jackson, MS (↗

DOE OSTI2024

Enhancement on selenium volatilization for phytoremediation: role of plant and soil microbe interaction

This study aimed at quantifying the potential effects of plant and soil microbial interaction on selenium (Se) volatilization, with the specific objectives of identifying soil bacteria associated with rabbitfoot grass (Polypogon monspeliensis) and demonstrating the enhancement of Se volatilization in the soil-Indian mustard (Brassica juncea) system through inoculation of the soil with the identified best Se-volatilizing bacterial strain. Soil bacteria were isolated from topsoil and rhizosphere soils of rabbitfoot grass, and the bacterial colonies were characterized via PCR-DGGE and DGGE band analysis prior to their identification using 16S rDNA sequencing technique.Bacillus cereusproduced over 500-fold more volatile Se in a culture medium treated with 15 µg Se/mL (equal mixture of SeO 4 2- , SeO 3 2- and selenomethionine) than any of the other eight identified bacterial strains. Inoculation of Indian mustard vegetated soil with the best Se volatilizing bacterial strainB. cereusresulted in a significant (p<0.05) increase in Se volatilization during a 7-day time period, compared to the soil-plant system without inoculation ofB. cereus. Thus, inoculation of the soil withB. cereussubstantially enhanced Se removal via biogenic volatilization in the soil-Indian mustard system. This study evaluated the role ofB. cereusin enhancing Se volatilization in soil-plant systems, and demonstrated the importance of plant and soil microbial interaction for Se phytoremediation.

Plant Sciences↗

DOE OSTI2025

Circular Perspective for Utilization of Industrial Wastewaters via Phytoremediation

Wastewater generated in municipal rendering facilities requires multi-step treatment, but it may also serve as a source of nutrients and water and thus may be valorized before or instead of the necessary wastewater treatment operations. In this work, wastewaters from a composting plant were utilized to support the growth of Miscanthus x giganteus, known as both a remediation plant and an energy biomass source. A pot experiment was established to compare the effects of different wastewater doses (0, 50, 100, and 200 mL per pot per week) on the miscanthus biomass yield, phytoextraction of heavy metals, biomass heat of combustion, and plant condition. The increase in the wastewater dose resulted in increases in both biomass yield (from about 44 to 139%) and biomass heat of combustion (from 7 to 17%) when compared to the control sample, with no adverse effects on plant physiological parameters. The highest concentrations of metals were found in miscanthus grown with the highest dose of wastewaters. It was found that higher wastewater dose correlates to both higher phytoextraction and phytorecovery of metals from plant substrate and wastewaters. The highest metal uptake was identified for Fe (431 mg·pot −1 ), followed by Al, Zn, Mn, Cu, Ni, Cr. The lowest metal uptake was noted for Pb, Co and Cd (0.88, 0.11, and 0.95 mg·pot −1 , respectively). The results indicate that miscanthus can be recommended for industrial wastewater treatment. In addition, due to high absorption efficiency of the substrate components, miscanthus can be used as a remediation tool, e.g., for the ecological stabilization of remediation of metal-polluted soils, especially in municipal facilities like rendering plants. This presents a circular perspective for the valorization of post-fermentation wastewaters with subsequent growth of energy crops, with other potential benefits for the environment, such as soil treatment, absorption of CO 2 , and air purification.

Miscanthus x giganteus↗

NASA NTRSDate not supplied

Biofilm Mitigation in Spaceflight Utilizing Phytoremediation

Explore the source record for details and available documents.

Duckweed↗

NASA NTRS2012

Phytoremediation of Indoor Air: NASA, Bill Wolverton, and the Development of an Industry

It was during this period of the early 1970's and 1980's when the issues associated with Sick Building Syndrome were gaining attention that the United States National Aeronautics and Space Administration (NASA) became an unlikely leader in identifying biological solutions to the problem of poor indoor air quality. NASA had been supporting work using biological systems for atmospheric regeneration since the 1950's, with the emphasis on using photosynthetic systems for the removal of carbon dioxide and regeneration of oxygen as part of a life support system. The then Soviet Union was conducting tests using algae systems in the BIO-1 program (1964-1968) to regenerate the air at the Siberian Branch of the Soviet Academy of Sciences in Krasnoyarsk (Later renamed the Institute of Biophysics). These tests were expanded to include the use of higher plants in the BIOS-2 testing in the 1970's, and humans during BIO-3 in the 1980'SI3. Within NASA, large scale testing of bioregenerative life support systems was conducted in the Biomass Production Chamber (BPC) at Kennedy Space Center, Florida as part of the Controlled Ecological Life Support Systems (CELSS) Breadboard project.

Stutte, Gary W.↗

DOE OSTI2021

Optimally-controlled hybrid phytoremediation system

A method for treating chemicals below ground is provided. The system includes contacting the chemicals with a rhizosphere to maximize transpiration rate and minimize over-saturation of the rhizosphere. Also provided is a method for using contaminated groundwater as a sole source of irrigation. The method uses extracting the contaminated water and pumping the extracted water to an irrigation zone under the control of an autonomous irrigation system. Each zone includes some trees and the root system for each tree is modified to function as a treatment cell. The invention also provides a system for treating contaminated water, using a contaminated water source situated below ground surface. The system uses a pump for extracting contaminated water from the water source and prevents the contaminated water from directly contacting the atmosphere above the ground surface. Vegetation which defines an underground rhizosphere is adapted to directly receive the water.

Yan, Y. Eugene↗

NASA NTRSDate not supplied

Update to Developing a Multi-bioreactor Test Stand for the Evaluation of Biofilm Mitigation Technologies

Biofilm formation in spacecraft wastewater systems is a complex and impactful problem. Biomass flowing in these wastewater systems can cause clogging, higher differential pressures, and flow restrictions impacting equipment reliability and function. To explore this issue, the Marshall Space Flight Center’s biofilm test stand was created and has been utilized to examine multiple biofilm mitigation techniques over a long duration test time. This paper is an update to a previous paper, ICES-2021-122, and will serve to discuss the modifications and enhancements to the biofilm test stand. While the previous configuration tested heat treatments and coatings, this phase of testing will examine differing materials, competing bacteria provided by an external bioreactor, a prototype phytoremediation unit, and additional testing with a nutrient filter. Configuration changes include the addition of float switches, adjustment of the drain assembly architecture and additional support hardware for the competing bacteria bioreactor and phytoremediation units.

ECLSS↗

NASA NTRSDate not supplied

Update to Developing a Multi-bioreactor Test Stand for the Evaluation of Biofilm Mitigation Technologies

ECLSS↗

NASA NTRSDate not supplied

Update to Developing a Multi-bioreactor Test Stand for the Evaluation of Biofilm Mitigation Technologies

ECLSS↗

DOE OSTI2022

Changing bioavailability of per- and polyfluoroalkyl substances ($\mathrm{PFAS}$) to plant in biosolids amended soil through stabilization or mobilization

Biosolids containing per- and polyfluoroalkyl substances (PFAS) could contaminate the receiving environments once they are land applied. In this report, we evaluated the feasibility of controlling the bioavailability of PFAS in biosolids to timothy-grass through stabilization or mobilization approaches. Stabilization was accomplished by adding a sorbent (i.e. granular activated carbon (GAC), RemBind, biochar) to biosolids, while mobilization was achieved by adding a surfactant, sodium dodecyl sulphate (SDS), to biosolids. The results showed that the ΣPFAS concentration in grass shoots grown in biosolids amended soil treated by GAC or RemBind at 2% was only 2.77% and 3.35% of the ΣPFAS concentration detected in shoots grown in biosolids amended soil without a sorbent, respectively, indicating the effectiveness of GAC and RemBind for stabilizing PFAS and reduce their bioavailability. On the other hand, mobilization by adding SDS to biosolids at a dose range of 10–100 mg/kg significantly increased the plant uptake of ΣPFAS by 15.48%–108.57%. Thus, mobilization by adding SDS could be a valuable approach for enhancing the PFAS removal if phytoremediation is applied. Moreover, higher rate of PFAS uptake took place after grass cutting was observed in this study. Thus, proper mowing and regrowth of timothy-grass could lead to efficient and cost-effective removal of PFAS from biosolids amended soil through phytoremediation and leave the site clean to be used for other purposes.

54 ENVIRONMENTAL SCIENCES↗

DOE OSTI2024

Comparison of mercury (Hg) bioaccumulation with mono- and mixed Lemna minor and Spirodela polyrhiza cultures

Mercury (Hg) is a prevalent and harmful contaminant that persists in the environment. For phytoremediation, it is important to discover which plants can bioaccumulate meaningful amounts of Hg while also tolerating its toxicity. Additionally, increasing biodiversity could create a more resilient and self-sustaining system for remediation. This study explores whether mixed populations of Lemna minor and Spirodela polyrhiza can better bioaccumulate and tolerate Hg than monocultures. Mono- and mixed cultures of L. minor and S. polyrhiza were grown in mesocosms of 0.5 μg/L or 100 μg/L Hg ( HgCl2) spiked water for 96 h. Change in weight of duckweed was used to assess Hg tolerance. Diffusive gradients in thin-films (DGTs) were used as surrogate monitoring devices for bioavailable levels of Hg. For biomass growth, the mixed culture of the L. minor was greater than the monoculture at the high dose. The L. minor accumulated more Hg in the mixed culture at the low dose while the S. polyrhiza was higher in the mixed at the high dose. Hg speciation in water was modeled using Windermere Humic Aqueous Model 7 (WHAM7) to compare the bioavailable species indicated by the DGTs. Potentially due to the controlled conditions, the WHAM7 output of bioavailable Hg was almost 1:1 to that estimated by the DGTs, indicating good predictive capability of geochemical modeling and passive sampler DGT on metal bioavailability. Altogether, the mixed cultures statistically performed as well as or better than the monocultures when tolerating and bioaccumulating Hg. However, there needs to be further work to see if the significant differences translate into practical differences worth the extra resources to maintain multiple species.

63 RADIATION, THERMAL, AND OTHER ENVIRON. POLLUTAN↗

DOE OSTI2023

Influence of irrigation approaches and spatial geolocation on tritium speciation, uptake and depuration

Pine needles and tree cores from a tritium (T) contaminated phytoremediation forest at the Savannah River Site (SRS in Aiken, SC) Mixed Waste Management Facility (MWMF) were measured for total T and T speciation and compared to other locations at the SRS and the surrounding area. Tree core ages ranged from 9 to 14 years old, covering over half of the ~20-year on-going remediation efforts, while pine needles represent more recent time periods of 1-to-2-year increments. Remedial irrigation efforts at the MWMF are found to directly influence the pine needle T concentrations. The T content in the MWMF samples is higher than non-irrigated needle samples from other locations around the SRS. Furthermore, the different forms of organic bound T are preferentially stored in tree core tissue, compared to pine needles where tritiated water dominates.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

DOE OSTI2020

Transporters and ascorbate–glutathione metabolism for differential cadmium accumulation and tolerance in two contrasting willow genotypes

Salix matsudana Koidz is a low cadmium (Cd)-accumulating willow, whereas its cultivated variety, Salix matsudana var. matsudana f. umbraculifera Rehd., is a high Cd-accumulating and tolerant willow (HCW). The physiological and molecular mechanisms underlying differential Cd accumulation and tolerance in the two Salix species are poorly understood. In this study, we confirmed that the differential Cd translocation capacity from roots to the shoots leads to the differential Cd accumulation in their aboveground parts between these two willow genotypes. Cadmium accumulation happens preferentially in the transport pathway, and Cd is mainly located in the vacuolar, cell wall and intercellular space in HCW bark by cadmium location analysis at tissue and subcellular levels. Comparative transcriptome analysis revealed that higher expressions of several metal transporter genes (ATP-binding cassette transporters, K + transporters/channels, yellow stripe-like proteins, zinc-regulated transporter/iron-regulated transporter-like proteins, etc.) are involved in root uptake and translocation capacity in HCW; meanwhile, ascorbate–glutathione metabolic pathways play essential roles in Cd detoxification and higher tolerance of the Cd-accumulator HCW. These results lay the foundation for further understanding the molecular mechanisms of Cd accumulation in woody plants and provide new insights into molecular-assisted-screening woody plant varieties for phytoremediation.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2024

Commentary: Duckweeds as model organisms for metabolic studies

Duckweeds have many practical applications, for example in human nutrition, as animal feed, in the production of bioplastics or vaccines, and phytoremediation (Acosta et al., 2021). Under most conditions, they reproduce asexually which provides genetically uniform material with predictable patterns of growth that make them ideal as sentinel organisms for phytotoxicity testing (Park et al., 2021). Asexual growth also results in high biomass production which makes duckweeds promising candidates as biofuel feedstocks (Acosta et al., 2021; Liang et al., 2023). In addition, duckweed species like Lemna minor and Spirodela polyrhiza are also reemerging as model organisms in plant biology as high-quality full genome assemblies and other genomic resources become available (Chang et al., 2016; Acosta et al., 2021). We argue that duckweed species are particularly of interest for the study of primary plant metabolism. Primary metabolism concerns the part of metabolism that is directly involved in the growth and development of plants, and which tends to be highly conserved among plant species. What makes duckweeds particularly attractive is that when grown on liquid media more precise control of physiological conditions can be attained relative to growth of plants in soil. Also, due to their relatively simple anatomical structure and asexual reproduction of fronds by budding, precise characterization of the physiological state under study is possible through one simple metric, i.e., the specific growth rate (rate of dry weight increase per existing dry weight), which can be incorporated relatively easily into metabolic models. This is not possible for land plants, such as Arabidopsis, because over the course of their life cycle, they go through multiple growth stages and phases of anatomical differentiation, which are much more complex to quantify. Furthermore, duckweeds can grow on organic substrates under heterotrophic or photomixotrophic conditions that facilitate isotope tracer studies. For example, in a previous study on duckweed by one of the authors, Lemna gibba (L). was grown on glucose with a position-specific 13 C-label that can be detected and resolved by Mass Spectrometry or Nuclear Magnetic Resonance spectrometry. Using this approach, the 13 C-label was traced into biomass compounds formed from glucose, particularly isoprenoid compounds. Some of the resulting labeling patterns were in apparent disagreement with predictions based on known metabolic pathways for the biosynthesis of isopentenyl pyrophosphate, the universal building block for isoprenoids, (Lichtenthaler et al., 1997). From this data it was deduced that isoprenoid compounds such as carotenoids and isoprenoid chains of phytol and plastoquinone, synthesized in the chloroplast, are produced via a previously unreported plant metabolic pathway, now known as the methylerythitol/deoxyxylulose-5-phosphate pathway (Lichtenthaler et al., 1997).

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2025

EMSL Community Science Campaign Meeting: Critical Minerals and Materials - Rhizo Critical Campaign Breakout Session Report Summary

The “Critical Minerals Biogeochemistry in the Rhizosphere – Ultramafic Soils (Rhizo Critical)” campaign breakout (BO) session was organized to identify major knowledge gaps and fundamental research needs in rhizosphere microbiology and geochemistry that, if addressed, could transform our ability to recover critical minerals from ultramafic soil systems. We sought to identify significant challenges that must be surmounted in the pursuit of deeper science knowledge. Our ultimate goal is to understand this landscape well enough to identify and prioritize opportunities for EMSL to make the greatest impact with Environmental Transformations and Interactions (ETI) science area research campaigns focused on the biogeochemical processes controlling the behavior of critical minerals and materials in the rhizosphere. The increasing demand for critical materials and minerals (CMM) in the U.S. has heightened interest in low-grade ores with much attention on ultramafic soils, which contain valuable metals such as nickel (Ni), chromium (Cr), manganese, cobalt (Co), and copper (Lee et al., 2025; DOE CMM Report, 2023) used in advanced battery, magnet, wiring and wind turbines, and stainless steel technologies. Metal hyperaccumulating plants grown in ultramafic soils can extract economically valuable concentrations of CMMs through the process of phytomining. This technology has evolved from phytoremediation, which involves using plants to cleanse contaminated environments by removing, detoxifying, or stabilizing pollutants like metals and organic compounds. Hyperaccumulator plants are capable of storing metals in their living tissues at concentrations hundreds to thousands of times higher than those found in 'normal' plants. For instance, while the average concentration of Ni in the dry matter of plants growing in typical soils is usually less than 5 µg g?¹, Ni hyperaccumulation is defined by concentrations exceeding 1,000 µg g?¹ (Corzo Remigio et al., 2020; Reeves et al., 2018). Phytomining research has primarily focused on Ni (Rylott and van der Ent, 2025), for which the U.S. has very limited conventional mines in operation. Most soils typically contain Ni concentrations ranging from 7 to 50 mg kg-1, whereas serpentine soils exhibit significantly higher levels, with Ni content often ranging between 700 and 8,000 mg kg-1 (Sobczyk et al., 2017). While more than 500 plant species in over 50 different families have been identified as Ni hyperaccumulators (Kidd et al., 2018), Ni phytomining (and phytominng in general) remains largely untested because most studies are short-term, small-scale, and conducted under simplified or artificially enriched conditions, so they fail to capture the low metal concentrations, environmental variability, and management constraints that would be needed for a field-scale demonstration. Few hyperaccumulator species have been validated as true “metal crops,” and their biomass production, stress tolerance, and rooting characteristics are usually too poor to yield economically meaningful metal outputs. Critically, the basic mechanisms of metal uptake, transport, and sequestration, especially as shaped by belowground processes such as root exudation, rhizosphere chemistry, and root–microbe interactions that control metal mobility and bioavailability (Montreemuk et al., 2023; Kidd et al., 2018; Durand et al., 2023; Alford et al., 2010), are still only partially understood, and downstream metal recovery from biomass is rarely optimized. Because these limitations stem from gaps in fundamental knowledge rather than from a failure of the concept itself (Rylott and van der Ent, 2025; van der Ent et al., 2015), there is a strong need for basic science that dissects plant metal homeostasis, rhizosphere and microbial processes, and their integration with soil chemistry and process engineering to design more robust, scalable phytomining systems.

Ahkami, Amirhossein↗

DOE OSTI2022

Genome-Wide Association Study Reveals Complex Genetic Architecture of Cadmium and Mercury Accumulation and Tolerance Traits in Medicago truncatula

Heavy metals are an increasing problem due to contamination from human sources that and can enter the food chain by being taken up by plants. Understanding the genetic basis of accumulation and tolerance in plants is important for reducing the uptake of toxic metals in crops and crop relatives, as well as for removing heavy metals from soils by means of phytoremediation. Following exposure of Medicago truncatula seedlings to cadmium (Cd) and mercury (Hg), we conducted a genome-wide association study using relative root growth (RRG) and leaf accumulation measurements. Cd and Hg accumulation and RRG had heritability ranging 0.44 – 0.72 indicating high genetic diversity for these traits. The Cd and Hg trait associations were broadly distributed throughout the genome, indicated the traits are polygenic and involve several quantitative loci. For all traits, candidate genes included several membrane associated ATP-binding cassette transporters, P-type ATPase transporters, oxidative stress response genes, and stress related UDP-glycosyltransferases. The P-type ATPase transporters and ATP-binding cassette protein-families have roles in vacuole transport of heavy metals, and our findings support their wide use in physiological plant responses to heavy metals and abiotic stresses. We also found associations between Cd RRG with the genes CAX3 and PDR3, two linked adjacent genes, and leaf accumulation of Hg associated with the genes NRAMP6 and CAX9. When plant genotypes with the most extreme phenotypes were compared, we found significant divergence in genomic regions using population genomics methods that contained metal transport and stress response gene ontologies. Several of these genomic regions show high linkage disequilibrium (LD) among candidate genes suggesting they have evolved together. Minor allele frequency (MAF) and effect size of the most significant SNPs was negatively correlated with large effect alleles being most rare. This is consistent with purifying selection against alleles that increase toxicity and abiotic stress. Conversely, the alleles with large affect that had higher frequencies that were associated with the exclusion of Cd and Hg. Overall, macroevolutionary conservation of heavy metal and stress response genes is important for improvement of forage crops by harnessing wild genetic variants in gene banks such as the Medicago HapMap collection.

59 BASIC BIOLOGICAL SCIENCES↗