Phytoremediation and P hytosensing of Explosives: The T arget genes

Phytoremediation and Phytosensing of

Explosives: The Target genesBy: Ariel Moore

BackgroundPlant metabolism of xenobiotics has three phases

Activation (transformation)

Conjugation

Compartmentation (elimination)

Need to KnowTNT

2, 4, 6-trinitortoluene

RDXHexahydro- 1, 3, 5-trinitro-1, 3, 5-triazine

Toxic, mutagenic, and carcinogenic

PhytoremediationCost effective and soil stays in place causing less ecosystem

disruption

Phytosensing

Structures

TNT RDX

Materials and MethodsArabidopsis 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 medium0.5 RDX2.0 TNTProportional amounts of DMSO

Total RNA extracted from all three growthsPurifiedAssessed with microarrays and RT-PCR

AnalysisTwo-Color Microarray

Only for RDXThree biological replicates and dye

swap technique

Affymetrix MicroarraySame biological total RNA as two-color

to prepare cDNA24,000 genes and 22,500 probe setsGuanine cytosine robust multi-array

analysis algorithm and logs

Analysis IIAmplification

Real-Time RT-PCR

Enzyme activation 2 min. 55 degrees Celsius

Denaturation 15 min 95 degrees Celsius

40 cycles of 15 seconds, 95 degrees C30 seconds, 55 degrees C30 seconds, 72 degrees C

Negative control without revers transcriptase

PCR products confirmed for size

Results

Two-Color ResultsMost highly upregulated

Leucine-rich repeat family protein (At4g33970)

Other upregulatedProtease inhibitor/lipid transferPutative mannitol transporterXyloglucan/xyloglucosyl transferase (At4g14130)Lipozygenase (LOX2;At3g45140)

Downregulated genesPutative cysteine proteasePutative protease inhibiorXyloglucan/xyloglucosyl transferase (At5g57560)

Affymetric ResultsMost highly upregulated

Lipozygenase (LOX2;At3g45140)

Other upregulated genesABC transporterUridine diphosphate transferase family proteinPutative peroxidase

Most highly downregulatedUnknown expressed protein

Other down regulatedGene encoding for neurofilament protein-relatedCYTOCHROME P450

Real Time RT-PCRCarried 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 genes28% unknown function27% transcription factor activity12% transferase activity8% transporter activity11% protein binding

DiscussionDetoxification of RDX

Several genes induced by RDXGenes upregulated (transferase and transporter)9 unknown genes in two-color and 20 from Affymetrix

Detoxification of TNTPlants readily uptake and accumulate TNT in the rootsGenes upregulated (transferase and transporter activities)30 unknown proteins functions upregulated

CommonalityFew significant genes were upregulated or downregluated in

both explosives.

SpeculationDiffer 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

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