Transcript Poster

Phytotransformation Pathways of the Energetic Material TNT
Murali Subramanian 1, Hangsik Moon2, Sarah Rollo1, David Oliver 2, Jacqueline V Shanks1
1Dept.
of Chemical Engineering; 2Dept. of Genetics, Development and Cell Biology, Iowa State University, Ames, Iowa
Pathway Studies
Basics
(TNT)
(2,2' Azo)
Abstract: Plants have shown the capacity to take up and
transform TNT, amongst other energetic materials, in the lab
and at a field scale. Axenic (microbe-free) plant systems used
in these studies have elucidated the unique role of plants in
TNT transformation. Based on numerous experiments and
analytical compound identification, a pathway depicting TNT
transformation has been developed. The pathway conforms
to a “green-liver” mode of xenobiotic transformation. C.
roseus and Arabidopsis have been studied extensively for
TNT transformation metabolite formation and kinetics.
Hydroxylamines have been shown to form as the primary
metabolites, capable of shifting the transformation pathway
in various directions. Radiolabeled 14C TNT-feed studies in
these systems show the eventual binding of a modified
parent molecule to the plant, thereby limiting its
bioavailability. Recent studies have concentrated on
determining the genes and enzymes involved in TNT
transformation by screening mutant populations.
Background:
With
over
40
explosive
(specifically
2,4,6trinitrotoluene)
contaminated
sites in the US, and many more
worldwide, there exists, in these
areas, a real potential for
ecosystem
damage
and
groundwater
contamination.
Phytoremediation,
broadly
defined as the application of
plants of plant-biomass to clean
up pollutant wastes (Figure 1), is
an inexpensive, self-sustaining,
ecologically
harmonious
treatment technology that may be
suitable
for
prevention
of
contamination.
Figure 1:Phenomena included in phytoremediation:
Phytoextraction (uptake and direct concentration),
phytodegradation (uptake and biological transformation),
phytovolatization, phytostabilization (immobilization of
the contaminant in the soil) and rhizodegradation (action
of microbes in the rhizosphere).
TNT Transformation Studies
•Pathway Structure
•Final fates of TNT and metabolites
•Residual Toxicity
Genetic Analyses
•Isolating efficient natural mutants
and generating hybrids
+
O2N
N
N
Figure 2: While field-scale studies are very significant,
basic metabolism-scale studies are important too as
they help isolate natural mutants and generate hybrids
to efficiently remove wastes. In addition, the toxicity
and end-points of phytoremediation are fully
understood.
N
NO2
H3 C
NO 2
CH3
NO2
NO2
NO2
CHO
O2N
(4ADNB)
NHOCOCH3
CH3
(2HADNT)
O2N
O2N
(4HADNT) CH
3
O2N
NHOH
NO2
NO2
NO 2
NHOCOCH3
Reduction
binuclear
metabolites
NO2
(2HDNT)
Oxidation
(2ADNT)
CH 3
3
NO2
O2 N
NO 2
NO 2
NHOH
(4ADNT) CH
NH2
O2N
OH
O2N
Abiotic Dimerization
CH3
CH3
(4HDNT)
NO2
O2N
OH
NH 2
Figure 6. Screening of Arabidopsis mutants to isolate
strains with higher resistance to TNT. Contrast in
germination of wild-type and mutants at 25 mg/L TNT,
three weeks after plating. The mutant shows near
complete germination, while the wild-type shows none.
(2ADNBA)
(2HDNBA)
OH
O2N
Phase II
Conjugation
COOH
CH 2OH
NH2
O2 N
NO2
NO2
(TNT-2)
CH 3
CH 3
NH-R3
O2N
Phase III
Incorporation into
biomass
(2A-1)
NH-R4
O2N
(4A-1)
CH3
CH3
(TNT-1)
NO2
O2N
NO2
O2N
NO2
NO2
NH-R1
NH-R2
BOUNDS
Figure 3: A proposed TNT transformation pathway describes the uptake and
transformation of TNT to various metabolites and conjugates (adapted from
Subramanian and Shanks, in “Phytoremediation”, edited by Steve McCutcheon
and Jerry Schnoor, p389-408). TNT is completely removed from the system within
120 hours. A combination of HPLC-PDA, MS and NMR were used to identify the
structure of the metabolites formed. This pathway, based on the “green liver
model”, shows the initial reduction of TNT to the hydroxylamines- 2hydroxylamine-4,6-dinitrotoluene and its isomer the 4-hydroxylamine-2,6,dinitrotoluene. The hydroxylamines are subsequently completely reduced to the
monoamines- 2-amino-4,6-dinitrotoluene and 4-amino-2,6,-dinitrotoluene.
Alternatively, the hydroxylamines can also be oxidized or isomerized to other
metabolite branches. These reduced, oxidized and isomerized metabolites are
then subjected to a plant conjugation mechanism, wherein plant biomolecules like
sugars are attached to their functional groups. These transformation steps serve
to reduce the toxicity of the parent TNT and polarize the compound. The
conjugated metabolites are then polymerized by the plant enzymes and attached to
the plant biomass, often irreversibly. Since these compounds have a final fate of
being “bound” to the plant, they are not immediately bioavailable in the
ecosystem.
TNT profile in Arabidopsis, extracellular
Figure 7. Comparison of TNT removal capacities of
wild-type and mutant Arabidopsis seedlings. 110 mg/L
of initial TNT killed the wild-type seedlings, but the
mutant did not die. Significant phytotoxic effects were
observed on the mutant, however. Metabolite and
genetic analysis on the mutant can potentially reveal
the genes and enzymes in TNT phytotransformation.
RB
Chromosome 1
Figure 8. An example of a map of the T-DNA insertion in an
enhancer trap mutant line. T-DNA was inserted in the 4th
exon of the gene in chromosome 1 in this mutant. The
arrow indicates the orientation of the gene, and ATG and
TAA are the start and stop codons of the gene, respectively.
TNT
2ADNT
4ADNT
Bounds
0.8
14
TNT, Fraction of initial
C (Total Carbon)
1
0.8
[TNT]in=19 mg/L
0.6
[TNT]in=30 mg/L
[TNT]in=42 mg/L
0.4
[TNT]in=58 mg/L
[TNT]in=110 mg/L
0.2
[TNT]in=125 mg/L
0.0
Heat-Killed Control
Evap+ Photodeg Control
Evaporation Control
0
20
40
60
80
Time (hours)
100
120
140
Figure 4: TNT transformation by 2-week old
axenic Arabidopsis seedlings. Extracellular
levels of TNT fall rapidly at low to medium
concentrations of TNT feed. At higher
concentrations, phytotoxic effects exerted by
TNT prevent its removal from the system.
GTA
Gene : At1g49560, myb family transcription factor.
14
1.2
LB
AAT
1.4
1.0
Efficient and commercially
viable phytoremediative
processes
N
CHO
(2ADNB)
Phase I
+
NO2 O2N
1.2
•Genetic and enzymatic analysis
Knowledge-base of genes, enzymes, metabolites
and kinetics involved in TNT phytotransformation
NO2 O2N
CH3 H3C
NO2
(4,4' Azo)
O-
CH3
Mole fraction (initial TNT or C label)
Metabolism Analyses
O-
Genetic Studies
Conjugates
Unknowns
0.6
0.4
0.2
Acknowledgements:
0
0
10
20
30
40
50
60
70
80
Time (hours)
Figure 5: A complete mass balance for TNT
transformation by C. roseus hairy roots,
wherein uniformly labeled 14C TNT was fed to
the roots, and the radioactivity levels in the
media and biomass were measured.
This research is supported in part by the U.S. Department of
Defense, through the Strategic Environmental Research and
Development Program (SERDP), Project CU-1319. The authors
would also like to acknowledge Dr. Beom-Seok Seo for his initial
efforts in the TNT screening procedure. The pathway figure
shown in this poster is a cumulative of work by many
researchers in this field.