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 TNTRDX

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 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

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

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