TRANSCRIPTIONAL PROFILING OF ARABIDOPSIS THALIANA ROOT RESPONSES TO MUNITIONS Drew R. Ekman3, W. Wal

1
TRANSCRIPTIONAL PROFILING OF ARABIDOPSIS THALIANA
ROOT RESPONSES TO MUNITIONSDrew R. Ekman3, W.
Walter Lorenz2, Alan E. Przybyla1, N. Lee Wolfe3,
Steven C. McCucteon3, Jeffrey F.D. Dean2 1
Department of Biochemistry and Molecular Biology,
University of Georgia, Athens, GA 30602, USA 2
Daniel B. Warnell School of Forest Resources,
University of Georgia, Athens, GA 30602, USA3
U.S. EPA, National Exposure Research Laboratory,
Ecosystems Research Division, Athens, GA 30605,
USA
Abstract   Serial Analysis of Gene Expression
(SAGE) was used to identify Arabidopsis thaliana
genes that respond to TNT and RDX exposure. Root
tissues from plants grown in sterile liquid
medium and exposed to sublethal amounts of the
two munitions were used to prepare SAGE
libraries, which were characterized to a depth of
approximately 30,000 tags each.
Transcriptome-level responses to the two
munitions were very different. The tag most
highly induced by TNT (27-fold greater than in
control tissues) represented a glutathione
S-transferase transcript, suggesting predominance
of a detoxification response. In contrast, the
tag most highly induced by RDX represented an
NPR1-like protein transcript, which may suggest
involvement of a more generalized stress
response. A large number of cytochrome P450
transcripts and an ABC transporter transcript
were strongly induced in the root tissues treated
with TNT, which strongly supports the
multiphase-phase model of xenobiotic metabolism
that has previously been proposed for plants
exposed to this compound. Other tags highly
induced by RDX, including those encoding
DNAJ-like proteins, vacuolar-processing enzyme,
and various transcription factors, clearly
demonstrate that different metabolic pathways are
brought to bear on these two munitions. To the
extent that it facilitates establishment of
plants on contaminated sites, better
understanding of the genes and pathways involved
in resistance and/or degradation of these
munitions by plants should help increase the
success of future phytoremediation strategies
directed at waste munitions and other
xenobiotics. This work was supported by a USEPA
NNEMS Graduate Fellowship to D.R.E. Although this
work was reviewed by EPA and approved for
presentation, it may not necessarily reflect
official Agency policy.

• Methods
•  
• Arabidopsis Sensitivity to Explosives
• Arabidopsis plants were grown in sterile liquid
  Murashige and Skoog medium for two weeks before
  dosing with different concentrations of TNT or
  RDX.
• Seedlings were inspected visually for obvious
  signs of toxicityspecifically leaf chlorosis and
  necrosis.
• Plants growing in 15 mg/L TNT or 150 mg/L RDX
  displayed a toxic response, but appeared to
  retain viability. These concentrations were
  chosen for the SAGE analyses.
•  
• Tissue Growth and Harvesting
• Plants were grown for two weeks before amending
  media with TNT (15 mg/L) or RDX (150 mg/L).
  Treatments were replicated in multiple flasks, as
  well as on different days.
• After 24 hours of exposure, plants were removed
  from the media and washed in dH2O.
• Plant roots were immediately excised and frozen
  in liquid nitrogen for storage prior to RNA
  extraction.
•  
• RNA Extraction and Creation of SAGE Libraries
• Total root RNA was isolated using LiCl
  precipitation of Chang et. al (4).
• SAGE libraries were made according to the SAGE
  Detailed Protocol, Version 1.0c (Velculescu et
  al., 1997a).

Conclusions   This SAGE study strongly supports
the multi-phase process proposed for plant
metabolism of TNT (5,6). In the first phase of
this process, cytochrome P-450 enzymes and mixed
function oxidases act to alter specific sites on
the toxic compound, making them more amenable to
conjugation with glutathione (GSH) or six-carbon
sugars in the second phase of detoxification
(Figure 2). These conjugation reactions are
generally catalyzed by glutathione S-transferases
(GST) and UDPglucosyltransferases. In the third
phase of detoxification the conjugated compound
is either sequestered in plant storage organelles
or secreted to the apoplasm. Finally, in the
final phase of processing, the conjugate is
rendered inactive through the addition of further
substitutions or by degradation processes.
  The gene expression differences noted between
the TNT and control SAGE libraries also point to
an oxidative stress response elicited by the
presence of toxic concentrations of TNT (Table
4). This stress may be caused by either the
parent molecule or any of its reactive, oxidized
derivatives. TNT is considered both a mutagen
and a carcinogen due in part to its tendency to
yield oxidative metabolites that can react and
couple with cytosolic or nuclear
components.   The Arabidopsis transcriptome
response to RDX reveals a drastically different
metabolic mechanism for dealing with this
explosive. In contrast to the TNT responses, the
SAGE results did not indicate major involvement
of oxidative stress enzymes and cytochrome P450s.
Thus, while metabolism of TNT in plants probably
requires the multiphase mechanism described
previously (Ekman et al. 2003), and as noted in
other plant species dosed with a variety of other
organic compounds, this may not be the case for
RDX. This will be a major consideration in the
engineering of plants for phytoremediation of
sites contaminated with RDX. For plants to be
able to remediate both of these explosives
simultaneously (both TNT and RDX are often found
together at polluted sites) it may be necessary
to introduce a suite of genes specific for
metabolizing each of these munitions.   Using the
insights gained from these studies, we hope to
provide a means by which more educated choices
can be made in the development of transgenic
plants and in the assessment of native plants for
suitability in remediation schemes.
Introduction   Over most of the last century,
manufacturing, processing, and storage of the
explosives, 2,4,6-trinitrotoluene (TNT) and
hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX),
have been responsible for extensive contamination
of soil, as well as ground and surface water,
throughout the U.S. and Europe. Unlike many
other organic compounds possessing nitro-
moieties, such as pesticides and various
feedstock chemicals, these explosives (Figure 1)
are highly resistant to biological degradation,
and are thus able to persist in the environment
for long periods of time. In recent years it has
been observed that certain plants have the
ability to remove TNT and RDX from their
surroundings, suggesting that plants may offer a
potential solution to the environmental
contamination problem presented by these
compounds. Unfortunately, few of the plants
species identified as having the ability to
extract munitions from soil are robust enough to
provide practical remediation of the extensive
contamination common to many of the sites known
today. In an attempt to address this limitation,
researchers have designed transgenic plants with
greatly enhanced abilities to tolerate and remove
TNT from their environment (1,2). However, our
current understanding of plant metabolism
underpinning the processes of uptake and
degradation of TNT and RDX is still rudimentary.
Further elucidation of these metabolic pathways
will facilitate specific engineering of plants
for improved removal and degradation of TNT and
RDX, as well as for tolerance and persistence in
environments where these compounds prove toxic
for normal plants. We have employed a functional
genomics approach using serial analysis of gene
expression (SAGE) (3) to identify the full range
of genes in the model plant, Arabidopsis
thaliana, that respond to TNT and RDX. Changes
in root gene expression were observed in
Arabidopsis plants grown in the presence of
sub-lethal levels of either TNT or RDX.
Identification of the genes responding to these
munitions will enhance our understanding of the
manner in which plants cope with TNT and RDX in
particular, and may extend to other xenobiotic
contaminants in the environment.
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