Transcript Phytoremediation and Phytosensing of Explosives: The Target genes

Phytoremediation and
Phytosensing of
Explosives: The Target
genes
By: Ariel Moore
Background
 Plant metabolism of xenobiotics has three phases
 Activation (transformation)
 Conjugation
 Compartmentation
(elimination)
Need to Know
 TNT
 2, 4, 6-trinitortoluene
 RDX
 Hexahydro- 1, 3, 5-trinitro-1, 3, 5-triazine
 Toxic, mutagenic, and carcinogenic
 Phytoremediation
 Cost effective and soil stays in place causing less ecosystem
disruption
 Phytosensing
Structures
TNT
RDX
Materials and Methods
 Arabidopsis thaliana
 Murashige and Skoog (MS) medium
supplemented
 RDX different milimolar concentrations
 TNT different micromolar
concentrations
 DMSO in proportion to the RDX
and TNT concentrations
RNA Preparation
 Grown on MS medium
 0.5 RDX
 2.0 TNT
 Proportional amounts of DMSO
 Total RNA extracted from all three growths
 Purified
 Assessed with microarrays and RT-PCR
Analysis
 Two-Color Microarray
 Only for RDX
 Three biological replicates and dye
swap technique
 Affymetrix Microarray
 Same biological total RNA as two-color
to prepare cDNA
 24,000 genes and 22,500 probe sets
 Guanine cytosine robust multi-array
analysis algorithm and logs
Analysis II
 Amplification
 Real-Time RT-PCR
 Enzyme activation 2 min. 55 degrees Celsius
 Denaturation 15 min 95 degrees Celsius
 40 cycles of
 15 seconds, 95 degrees C
 30 seconds, 55 degrees C
 30 seconds, 72 degrees C
 Negative control without revers transcriptase
 PCR products confirmed for size
Results
Two-Color Results
 Most highly upregulated
 Leucine-rich repeat family protein (At4g33970)
 Other upregulated
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

Protease inhibitor/lipid transfer
Putative mannitol transporter
Xyloglucan/xyloglucosyl transferase (At4g14130)
Lipozygenase (LOX2;At3g45140)
 Downregulated genes
 Putative cysteine protease
 Putative protease inhibior
 Xyloglucan/xyloglucosyl transferase (At5g57560)
Affymetric Results
 Most highly upregulated
 Lipozygenase (LOX2;At3g45140)
 Other upregulated genes
 ABC transporter
 Uridine diphosphate transferase family protein
 Putative peroxidase
 Most highly downregulated
 Unknown expressed protein
 Other down regulated
 Gene encoding for neurofilament protein-related
 CYTOCHROME P450
Real Time RT-PCR
 Carried out to confirm the microarray results
 6 genes were used
 Molecular functions of differentially expressed genes in
response to TNT and RDX were different.
 Functional categorization for upregulated genes





28% unknown function
27% transcription factor activity
12% transferase activity
8% transporter activity
11% protein binding
RDX two-color up-regulated genes
RDX two-color down-regulated genes
RDX Affymetrix up-regulated genes
RDX Affymetrix down-regulated genes
TNT Affymetrix up-regulated genes
TNT Affymetrix down-regulated genes
Discussion
 Detoxification of RDX
 Several genes induced by RDX
 Genes upregulated (transferase and transporter)
 9 unknown genes in two-color and 20 from Affymetrix
 Detoxification of TNT
 Plants readily uptake and accumulate TNT in the roots
 Genes upregulated (transferase and transporter activities)
 30 unknown proteins functions upregulated
Commonality
 Few significant genes were upregulated or downregluated in
both explosives.
 Speculation
 Differ chemically and have different metabolic pathways
Future Applications and
Studies
 Need phytoremediation of both compounds
 Identification of either TNT or RDX
 Characterizing the
identified genes
References
 "Landmines | Eric W. Barton - Man on a Mission." Eric W Barton Man on a Mission
Landmines Tag. N.p., n.d. Web. 05 Dec. 2012.
<http://www.ericbartononline.com/tag/landmines/>.
 Panz, Katarzyna, and Korneliusz Miksch. "Phytoremediation of Explosives (TNT, RDX,
HMX) by Wild-type and Transgenic Plants." Science Direct. N.p., n.d. Web. 28 Nov. 2012.
<http://www.sciencedirect.com/science/article/pii/S0301479712004240>.
 Rao, Murali, and Et. Al. "Phytoremediation and Phytosensing of Chemical Contaminants,
RDX and TNT: Identification of the Required Target Genes." Springer Linker. SpringerVerlag, 19 June 2009. Web. 29 Nov. 2012.
<http://link.springer.com/article/10.1007/s10142-009-0125-z/fulltext.html>.
 "Research Unit Microbe-Plant Interactions: Research » Research
Groups » Root Ecophysiology » Research." Research Unit Microbe-Plant
Interactions: Research » Research Groups » Root
Ecophysiology » Research. N.p., n.d. Web. 05 Dec. 2012. <http://www.helmholtzmuenchen.de/en/amp-eng/research/research-groups/rootecophysiology/research/index.html>.
 Rylott, Elizabeth L., and Neil Bruce. "Plants Disarm Soil: Engineering Plants for the
Phytoremediation of Explosives." Science Direct. N.p., Feb. 2009. Web. Nov. 28.
<http://www.sciencedirect.com/science/article/pii/S0167779908002874>.