Abstracts from the Joint Interagency Phytoremediation
Research Program
Genetic and Molecular Dissection of Arsenic Hyperaccumulation in the Fern Pteris vittata. Jo Ann Banks,
The goal of the proposed
research is to identify the genes that are necessary for arsenic hyperaccumulation in Pteris vittata using molecular and genetic
approaches. Specific objectives
include: 1) identifying P. vittata
genes that are involved in arsenic hyperaccumulation
by functional complementation in S. cerevisiae, 2) using a genetics approach to identify
natural variants or mutations of P. vittata genes that negatively affect the plant's
ability to tolerate or hyperaccumulate arsenic, and
3) identifying and characterizing arsenic tolerant mutants in Ceratopteris richardii, a
fern that is related to P. vittata and is sensitive to arsenic. For objective 1, the function of P. vittata
genes will be determined using a reverse genetics approach recently developed
for ferns, and then the appropriate genes will be overexpressed
in Arabidopsis to see whether
expression can confer arsenic tolereance. For objective 2, a mutagenesis and genetic
screening approach will be used to identify the genes and mechanisms underlying
arsenic hyperaccumulation in P. vittata. For objective 3, arsenic resistant and
accumulating Ceratopteris richardii
ferns will be identified and characterized to assist in providing genetic
information on the number and type of mutations needed to change an arsenic
non-accumulator into a hyperaccumulator.
DOE Project Officer: Paul E. Bayer
Project Period of
Performance:
Project Award Total: $450,000
Key Words: phytoremediation,
arsenic, ferns, Pteris vittata,
genes, molecular genetics, hyperaccumulation, Arabidopsis, Ceratopteris richardii
Enhancement of Selenium Volatilization by Salicornia: Plant and Microbial Interactions. Zhi-Qing Lin,
Southern
The proposed research aims
to elucidate and manipulate the important environmental and biological factors
that limit the high production rates of volatile selenium (Se). The following three hypotheses will be
examined: 1) microbial volatilization constitutes the most
significant and effective pathway of Se removal in the soil-Salicornia system, 2) Salicornia
provides special microbial habitats in support of the species-specific soil
microbial populations with accelerated ability for Se volatilization in the Salicornia field,
and 3) phytotransformation of inorganic to organic Se
and high availability of organic Se from the decomposition of plant biomass are
the key factors facilitating the process of microbial Se production in a
soil-plant system. Specific objectives
are: 1) to determine the respective
contribution of Salicornia
plant and soil microbes to the total Se volatilization in the soil-plant
system, 2) to identify Salicornia-associated
microbial populations with accelerated abilities for Se volatilization, and 3)
to explore the interaction between Salicornia and associated microbes and the mechanisms
underlying the enhancement of Se volatilization in the Salicornia phytoremediation
system.
DOE Project Officer: Paul E. Bayer
Project Period of
Performance:
Project Award Total: $81,951
Key Words: phytoremediation,
selenium, Salicornia,
soil microbiology, volatilization
A Phytoremediation Strategy
for Arsenic. Richard B. Meagher,
The proposed research will
develop a genetics-based phytoremediation strategy
for arsenic removal that can be used in any plant species. The working hypothesis is that organ-specific
expression of genes controlling the transport, electrochemical state, and
binding of arsenic will result in the efficient extraction and hyperaccummulation of arsenic into above ground plant
tissues. The hypothesis will be tested
through the following research objectives: 1) enhance plant resistance and
expand sinks for arsenite by expressing elevated
levels of thiol-rich arsenic-binding peptides; 2)
convert arsenate to arsenite in above ground organs
by expressing a bacterial arsenate reductase gene (ArsC) under a light mediated leaf promoter; 3) characterize
endogeneous root-specific arsenic reductase
(AtACR2) and enhance the transport of arsenate from roots to shoots by suppressing
the activity of the enzyme; 4) enhance arsenate uptake via overexpression
of high-affinity phosphate transporters; 5) enhance intracellular transport of thiol-arsenate complexes into vacuoles in leaf cells by
elevating the expression of a glutathione conjugate pump in above ground
organs; and 6) combine these transgenic elements into test plants (Arabidopsis and tobacco) and demonstrate
dramatic increases in arsenic resistance and hyperaccumulation. The general approach will be to initiate each
experiment in Arabidopsis and confirm
the most informative experiments in tobacco.
DOE Project Officer: Paul E. Bayer
Project Period of
Performance:
Project Award Total: $450,000
Key Words: phytoremediation,
arsenic, arsenate reductase, Arabidopsis, hyperaccumulation, molecular
genetics
Phytoremediation of Marine Sediments Contaminated with Polynuclear
Aromatic Hydrocarbons and Polychlorinated Biphenyls Using Eelgrass (Zostera marina). Michael Huesemann,
The proposed research will examine the effects of
eelgrass root zone aeration on biodegradation of PAH’s
and PCB’s. It is hypothesized that eelgrass will increase photosynthesis-driven
oxygen delivery to rhizosphere microbes and will
enhance aerobic degradation and increase the microbial biomass and diversity.
Using aquaria, the effects of eelgrass on the extent of PAH/PCB removal will be
studied as a function of sediment depth (and correlated with root zone depth).
In addition, microbial diversity and biomass will be enumerated as both
epiphytic and rhizosphere populations. Transfer of PAHs/PCBs into the water column will be measured, as will
uptake into eelgrass tissues. The rate of oxygen release into the rhizosphere will be measured over light and dark cycles to
ascertain the role of photosynthesis. These data will be used to
semi-quantitatively identify the magnitude of eelgrass-enhanced biodegradation
of PAHs and PCB’s.
ONR Project Officer: Linda Chrisey; DOE
Project Officer: Mr. Paul Bayer
Project Period of Performance:
Project Award Total: $493,926 (ONR) + $70,000 (DOE) =
$563,926
Key Words: Zostera
marina; eelgrass; phytoremediation; marine
sediments, rhizosphere, polyaromatic
hydrocarbons, polychlorinated biphenyls
Mechanistic Role of
Plant Root Exudates in the Phytoremediation of
Persistent Organic Pollutants. Jason White, MaryJane Incorvia Mattina, Martin Gent, Barth Smets, Daniel Gage, Connecticut Agricultural Station, University
of Connecticut
This proposal is designed to investigate the role
of plant root exudates in the phytoremediation of
persistent organic pollutants in soil. Preliminary data have shown that two
weathered organic pollutants (p,p'-DDE, chlordane) are readily translocated
from soil to the tissues of certain plants. These findings contradict a
significant body of scientific evidence indicating time-dependent reductions in
contaminant availability in soil (i.e., sequestration). We propose a novel
mechanism of phytoremediation whereby plant root
exudates increase the bioavailability of weathered contaminants. The following
hypotheses will be tested: (1) The
root exudates of certain plant species facilitate the mobility and subsequent
availability of weathered organic pollutants; and (2) Contaminant solubilization by exudates occurs by direct or indirect
mechanisms. In direct enhancement, the exudate
molecules directly induce contaminant release from the soil. Possible
mechanisms here include the formation of exudate/contaminant
complexes or the partial solubilization/reformation
of soil structure organic fractions through chelation
of polyvalent metals (iron and aluminum). A second hypothesis considers
indirect enhancement, where root exudates stimulate a microbial community that
promotes contaminant availability to the plant.
EPA Project Officer: Mitch
Lasat
Project Period
of Performance:
Project Award Total: $401,241
Key Words: phytoremediation, root exudates,
organic pollutants, soil microbiology
Evaluation of Monoterpene Producing Plants for Phytoremediation
of PCB and PAH Contaminated Soils. David Crowley, James Borneman,
University of California-Riverside
Plants produce a
variety of chemicals with structures that are analogous to those of many
commercially produced chemicals. Rhizodeposition of
these substances can beneficially affect xenobiotic
degradation by promoting selective enrichment of degrader organisms,
enhancement of growth-linked metabolism, and induction of genes for enzymes
that facilitate cometabolism. In previous research,
we have exploited the ability of plant monoterpenes
to induce bacteria to cometabolize PCBs. Data from
the literature and our prior research suggest that terpenes
produced in situ by plants also should be effective for promoting degradation
of many organic contaminants, including PAHs and
other recalcitrant contaminants. The objective of the proposed research is to
evaluate monoterpene-producing plant species for use
in phytoremediation of PCBs and PAHs,
and to investigate the ecology of indigenous xenobiotic
degrading bacteria in the rhizosphere of monoterpene producing plants. Experiments will test four
hypotheses: (1) the rhizosphere selectively enriches
for diverse populations of xenobiotic degrading
microorganisms that occur at higher population densities in the rhizosphere as compared to the bulk soil; (2) plant and
microbial substances that are released into the rhizosphere
enhance the expression and activity of inducible enzymes that work in concert
to degrade xenobiotic soil contaminants; (3) monoterpene producing plants selectively enrich for diverse
populations of xenobiotic degrading microorganisms
that will occur at higher population densities in the rhizosphere
as compared to the plants that do not produce these substances; and (4) plant
enhanced remediation of PAH and PCBs in the rhizosphere
can be enhanced by the addition of earthworms to improve soil aeration for
aerobic degradation processes.
EPA Project Officer: Mitch Lasat
Project Period of Performance:
Project Award Total:
$393,135
Key Words: phytoremediation, biodegradation, rhizosphere,
soil microbiology, polycyclic aromatic hydrocarbons, polychlorinated biphenyls,
monoterpenes
Title: Physiological Mechanisms
of Estuarine Sediment Oxidation by Spartina Cordgrasses. Raymond Lee,
Cordgrasses of the genus Spartina will be investigated for their potential use as a phytoremediation tool in marine and estuarine sediments. Spartina grasses
are adapted to saline, waterlogged sediments and exhibit vigorous growth,
forming dense monospecific stands in a variety of intertidal environments. The capability of these plants to
transport oxygen from the atmosphere to the belowground rhizosphere
has the potential to enhance microbial degradation of organic pollutants, which
can be limited by oxygen availability in anoxic waterlogged soils. The specific
objectives are as follows: (1) determine rates of oxygen transport and release by
Spartina
grasses; (2) identify species and strains of Spartina that have enhanced
oxygen release capabilities; (3) determine the mechanisms that facilitate
oxygen transport, and how transport is induced by environmental and hormonal
signals. These studies will assist in recovery of estuarine environments
affected by pollution.
EPA Project Officer: Mitch Lasat
Project Period of Performance:
Project Award Total:
$110,307
Key Words: phytoremediation, aquatic grasses,
Spartina, marine sediments, rhizosphere
The Molecular Basis for Heavy Metal Accumulation and
Tolerance in the
Hyperaccumulating Plant Species, Thlaspi caerulescens. Leon V. Kochian,
The goals of this research are to identify the basic mechanisms of heavy
metal hyperaccumulation in plants, and to isolate and characterize the suite of
genes that underly this hyperaccumulation trait in Thlaspi caerulescens. Dr. Kochian's group will use recent advances
in plant molecular biology and genomics to identify both metal transporter
genes involved in metal accumulation and tolerance, as well as genes involved
in the production of low molecular weight organic compounds (e.g., peptides,
organic genes, amino acids, metallothioneins, phytochelatins) that can bind and
detoxify Zn and Cd in plant cells. Based on the recent sequencing and analysis
of the Arabidopsis genome, it is now known that higher plants employ the same
families of metal transporters recently identified and characterized in yeast,
bacteria and mammals for metal accumulation and homeostasis. Dr. Kochian's
group has cloned genes in T. caerulescens
from these different metal transporter gene families and will characterize
these transporters to determine their role in metal hyperaccumulation. This
characterization will include determining in which plant tissue and cell type
different genes are expressed, the membrane localization of transport proteins
to help assign a potential role for each transporter, and the elucidation of
the physiological function of individual metal transporters. They also are
expressing T. caerulescens genes in
yeast to look for genes conferring metal tolerance through the production of
metal binding organic ligands.
These approaches should allow the investigators to identify the suite of
genes that confer heavy metal hyperaccumulation in T. caerulescens and to elucidate the molecular mechanism(s) for
this trait. The ultimate goal of this research is to use these
hyperaccumulation genes to develop transgenic plants that both are metal
hyperaccumulators and produce high shoot biomass , and thus will be well suited
for the phytoremediation of metal contaminated soils.
NSF Program Manager: William
E. Winner
Project Period:
Award Total: $416,927
Key Words: phytoremediation, heavy metals, Thlaspi, hyperaccumulator,
molecular genetics
Genome-Wide Hunt For Metal Hyperaccumulation Genes. David E. Salt,
The overall objective of
this project is to identify genes involved in metal hyperaccumulation
in metal-hyperaccumulating plants. These unique plant
species are able to accumulate between 0.1 and 3% of their shoot dry biomass as
Cd, Ni, Se or Zn depending on the species. Over 25%
of the known hyperaccumulator species are members of
the Brassicaceae family, and as such they are related
to Arabidopsis thaliana. By
investigating the molecular genetics of metal hyperaccumulation
in species related to A. thaliana,
the investigators will utilize the technical and genetic resources developed during
the Arabidopsis genome project,
harnessing powerful functional genomics technologies to dissect metal hyperaccumulation at the genetic level.
Metal hyperaccumulators
in the Brassicaceae will be collected from around the
world, and genes important in hyperaccumulation will
be identified using three complementary approaches. Seeds from approximately 40
accessions of over 20 different species of hyperaccumulators
in the Brassicaceae family will be collected from
NSF Program Manager: William
E. Winner
Project Period:
Award Amount: $450,000
Key Words: phytoremediation, metals, hyperaccumulator,
Arabidopsis, molecular genetics
Molecular Mechanism of Nickel
Hyperaccumulation in Thlaspi goesingense.
David E. Salt,
Intensive industrial and agricultural activity over the last 150 years has
imposed a large burden of heavy metals on the environment. Phytoextraction, the
use of plants for environmental cleanup of pollutants, including toxic metals,
from soils, holds the potential to allow the economic restoration of these
contaminated sites. For phytoextraction to be a viable alternative to existing
soil remediation strategies it will require the existence of high biomass,
rapidly growing metal-accumulating plants. Unfortunately, plants do not exist
at present that have all these desirable characteristics. There are, however, a
limited numbers of plants, collectively termed hyperaccumulators, that grow on
soils naturally enriched in various metals including Zn, Ni and Se. These
plants have the ability to naturally accumulate these metals to between 0.1 and
3% of their shoot dry weight; this is at least 1000-fold higher than most other
plants. This unique ability makes these plants an ideal starting point for the
development of phytoextraction crops. One way to develop such crops is to
identify the genes responsible for metal accumulation in these hyperaccumulator
plants. Once identified and fully characterized these genes could be
transferred into high biomass, rapidly-growing plants to generate crops ideally
suited for phytoremediation. This grant will fund the identification of such
"metal hyperaccumulation" genes from the nickel hyperaccumulator Thlaspi goesingense. Once identified,
the usefulness of these genes for phytoremediation will be rapidly assessed by
their transfer to Arabidopsis thaliana, a convenient model plant. Genes
identified for enhanced metal tolerance and accumulation in this model plant
will then be selected for transfer to plants more suited to phytoremediation
applications.
NSF Program Manager: Stephen
Herbert
Project Period:
Award Amount: $329,806
Key Words: phytoremediation, phytoextraction,
metals, hyperaccumulator, Thlaspi, molecular genetics
Intraspecific Variation in Thlaspi caerulescens: The Key to Increasing
Metal
Sequestration in Plants. Stephen D. Ebbs, Southern
Metal hyperaccumulation and hypertolerance are unique traits observed in a
limited variety of plant species from around the world. A relatively
understudied aspect with respect to metal hyperaccumulating plant species is
the natural variation in hyperaccumulation and tolerance observed between
populations within a species (i.e., intraspecific variation). For example,
recent studies have shown marked differences in the hyperaccumulation of Cd and
Zn across populations of Thlaspi
caerulescens. The inherent variability in hyperaccumulation displayed by T. caerulescens and the unique cellular
and subcellular patterns of metal distribution (e.g. - epidermal and vacuolar
sequestration) in leaves provide a natural model system in which to examine the
leaf-level mechanisms that control metal homeostasis. Understanding the basis
of this variation will contribute to the ongoing efforts to develop more
efficient hyperaccumulators for metal phytoremediation and metal
"bio-mining". To examine these leaf-level mechanisms, this project
will (1) use cell viability assays to compare the metal tolerance of leaf
mesophyll cells from different populations of T. caerulescens to determine the contribution of these cells to the
intraspecific variation in metal hyperaccumulation; (2) conduct radiotracer
transport studies with isolated vacuoles and/or vacuole membranes from
different populations to determine whether intraspecific variation in the rate
or extent of vacuolar sequestration contributes to hyperaccumulation; and (3)
use 2-D protein gel electrophoresis to determine if the more efficient
metal-accumulating plant populations possess novel proteins that contribute to
their ability to tolerate and sequester metals in leaves. Together the results
of this study will add to our understanding of the relationship between metal
distribution in leaves and the extent to which different populations of T. caerulescens hyperaccumulate Cd and
Zn. This unique insight into the metal dynamics in leaves will be instrumental
in the development of higher biomass plants for phytoremediation.
NSF Program Manager: William
E. Winner
Project Period:
Award Amount: $100,000
Key Words:phytoremediation, metals, hyperaccumlator,
Thlaspi, molecular biology
Are Plant Root-Mycobacterium Interactions Beneficial
in Remediation of
Polyaromatic Hydrocarbons? Anne J. Anderson,
PAH-contaminated soils pose environmental and health hazards.
Phytoremediation is a cost effective method for on-site clean-up. It is well
suited for large surface areas such as those designated as “brownfields” within
urban settings or sites where soil excavation and removal is difficult. This
proposal focuses on understanding more of the ecology of mycobacteria that have
PAH-degrading potential. Currently there is little knowledge of how such
mycobacteria interact with plant roots and whether this association has
positive impacts on the metabolism of the plant and/or the microbe to promote
bioremediation. The hypotheses to be
tested are: 1) The presence of roots colonized by PAH-degrading mycobacteria
improves the bioavailability of a model, recalcitrant PAH, pyrene; 2) The
mineralization of pyrene is enhanced by the interaction of the roots with the
mycobacteria; 3) Colonization of the root requires discrete interactions
between the mycobacteria and root surface; 4) Colonization of the root permits
the expression of the gene in mycobacteria encoding the first enzyme involved
in PAH degradation, dioxygenase; 5) Root phenoloxidases, which may participate
in PAH-remodeling, are changed in activity in the roots colonized by
mycobacteria.
NSF Program Manager: William
E. Winner
Project Period:
Award Amount: $398,336
Key Words: phytoremediation, plant-microbe interactions, polyaromatic hydrocarbons, mycobacteria
Molecular Genetics of Polycyclic Aromatic Hydrocarbon
Stress Responses and Remediation by Arabidopsis
thaliana. Adan Colon-Carmona,
The proposed project explores the underlying molecular mechanisms for polycyclic
aromatic hydrocarbon (PAH)-induced responses in plants, as well as their
potential biodegradation pathways. PAHs are organic pollutants that cause human
health problems such as cancer. PAHs are contaminants resulting from oil-based
manufacturing. Some plant species, including crop plants such as sunflower,
soybean, pea and carrot, can grow on moderate levels of crude oil-contaminated
soil. Yet, very little is known at the molecular level about the mechanisms of
PAH uptake and degradation, or even cell signaling pathways regulating PAH
stress responses. A better understanding of PAH stress physiology will lead to
the generation of phytoremediation strategies in pollution clean-up and
biomonitoring. The aims of this proposal are the following: 1) to characterized
the physiological responses to PAHs in Arabidopsis
thaliana, 2) to identify the signaling pathways that mediate the various
PAH-induced plant responses, 3) to screen genetically mutagenized populations
for plants that are defective in PAH-induced growth responses, and 4) to
identify, through bacterial screens, plant cDNAs that can be used in PAH
degradation. The long term goal of these studies are to utilize the information
regarding PAH-induced responses in Arabidopsis
to engineer trees or crop plants with extensive root systems for their use in
biodegradation and biomonitoring of PAH contamination.
NSF Program Manager: William
E. Winner
Project Period:
Award Amount: $340,000
Key Words: phytoremediation, polyaromatic hydrocarbons, Arabidopsis, molecular genetics
Understanding and Enhancement of Arsenic Hyperaccumulation by a Fern Plant. Jean-Francois Gaillard,
Northwestern University and
The objective of this research is to understand the mechanisms of arsenic
uptake, translocation, distribution and detoxification by Brake fern. The
efficiency of arsenic uptake by Brake fern suggests the cost-effective use of
this plant for the remediation of arsenic contaminated soils. This research
focuses on elemental interactions of arsenic with calcium and phosphorus, plant
biochemical responses under arsenic stresses, speciation and characterization
of arsenic in the plant using analytical, microscopic and spectroscopic
techniques, and microbe-root-plant-arsenic interactions. Arsenic
hyperaccumulation characteristics in Brake fern growing in soils of different
arsenic concentrations will be investigated using arsenic spiked soils. The
impacts of P (increases arsenic availability yet competes with arsenic uptake)
and Ca (increases plant arsenic uptake and translocation) on arsenic
accumulation, and biochemical responses of Brake fern to elevated arsenic
(detoxification) will be examined. Also, the beneficial effects of mycorrhizal
fungi for enhancing arsenic accumulation by Brake fern will be explored. This
is a collaborative research project between the University of Florida and
Northwestern University.
NSF Program Manager:
Nicholas Clesceri
Project Period:
Award Amount: $376,672
Award Amount: $358,000
Key Words: phytoremediation, arsenic, Brake fern, hyperaccumulator
Applications of 13C tracer studies and stable isotope
geochemistry to determine rhizosphere alteration of
PAH bioavailability in contaminated geomedia. Dr. Elizabeth G. Nichols,
This proposal uses polycyclic aromatic
hydrocarbons (PAHs) as model contaminants to
delineate how the rhizospheres of plant systems
impact weathered contaminant sequestration and bioavailability. We propose to
use two isotopic tracer approaches in which 13 CO2 photosynthetic labeled plant exudates or
uniformly labeled 13 C-PAHs are
introduced into weathered PAH contaminated media vegetated with Phragmites australis. Three field sites will be used to provide
PAH weathered geomedia. We will determine if organic
matter composition in geomedia fractions from the rhizosphere zone differs from
non-rhizosphere geomedia over time
and if compositional differences alter PAH
partitioning, desorption, and
toxicity in specific fractions such as particulate fractions and diagenetic fractions such as black carbon and humic materials.
NSF Project
Officer: Thomas Waite
Project
Period of Performance:
Project Award
Total: $434,103
Key Words: phytoremediation, rhizosphere,
PAH
Involvement of
an endosymbiotic Methylobacterium sp. in the biodegradation of explosive
RDX and HMX inside poplar tree (Populus deltoides). Dr. Jerald Schnoor,
The main objective of the
proposed research is to investigate the involvement
of endophytic pink pigmented
facultative methylotrophic (PPFM) bacteria in the
bioremediation of the RDX and HMX inside poplar trees. A secondary objective is to characterize the
symbiotic plant-bacteria relationship and the extent of poplar contamination by
PPFM bacteria. The hypothesis is that endosymbiotic
microbes, such as PPFM, living inside woody plants are involved in and can
improve significantly phytoremediation of organic
pollutants. Enhanced biodegradation originates either directly from bacterial
metabolism or from an improved plant metabolism due to symbiotic association
with the bacteria.
NSF Project
Officer: Thomas Waite
Project
Period of Performance:
Project Award
Total: $247,441
Key Words: phytoremediation, RDX, HMX, Populus, endosymbionts,
methylotrophs