Why is TNT toxic to plants?

(above, Arabidopsis plants growing in TNT-contaminated soil, the mutant plants are on the right-hand-side)

In our paper we show why TNT is toxic to plants

Monodehydroascorbate reductase mediates TNT toxicity in plants

Emily J. Johnston1,*, Elizabeth L. Rylott1,*,†, Emily Beynon1, Astrid Lorenz1, Victor Chechik2, Neil C. Bruce1,†

Science (2015) 349: 1072 -1075

http://www.sciencemag.org/content/349/6252/1072.abstract?sid=79d47c0a-5bbf-4b21-b9b3-6b4b1d02c3a4

Why is this important?

Knowing why TNT is toxic will enable us to develop plants that can be used to re-vegetate and detoxify land contaminated by TNT. It is difficult to estimate global levels of TNT contamination, many sites are unrecorded, or records lost, but as a marker, in the US it is estimated that there are about 10 million hectares of military land is contaminated with munitions constituents, many of which contain TNT. In Europe for example, there are many TNT manufacturing sites left over from the 1st and 2nd World War where TNT in the soil is not degrading. While some soil microbes have evolved resistance to TNT, and a few can even detoxify it, data indicate that TNT is not being degraded in soils, with contamination remaining for decades. For reasons scientists don't fully understand, microbes alone are insufficient to remediate TNT-contaminated soil.

What we did in the lab

We conducted a screen to find plants that were resistant to TNT. We used a model plant called Arabidopsis (Arabidopsis thaliana) and screened plants that had been chemically mutated.

Arabidopsis is a small, fast-growing plant enabled us to screen 1000s of plants quickly and in a small space. The genome of this plant is fully-sequenced and many of the genes have been characterised, these factors enabled us to locate the mutated gene and identify additional mutants from publicly available, mutated populations.

Seedlings were screened on plates of agar containing TNT. Once we isolated TNT-resistant mutants, we determined which gene was mutated. Using this method we isolated a TNT-resistant plant with a mutation in theMDHAR6 gene. To verify that MDHAR6 was conferring TNT-resistance, we restored an active (non-mutated) copy of MDHAR6 into the plant. This restored TNT-sensitivity. To understand the action that MDHAR6 was performing on the TNT molecule, we made and purified the protein and used a range of techniques to demonstrate the activity outlined below.

What does this MDHAR6 do?

The enzyme, MDHAR6 converts TNT to a more toxic compound which then reacts with oxygen to release free radicals. The free radicals damage the plants cellular machinery. During the reaction with oxygen, the original TNT molecule is restored, enabling MDHAR6 to repeat the 'toxic cycle'.

What next?

Arabidopsis could not be used to re-vegetate TNT-contaminated land- it is too small and weedy. We would need to identify the same mutation in more robust plants suitable for phytoremediation. Switchgrass and some other high-biomass species would be appropriate. The plants would not have to be genetically modified, we could use chemical mutagens, and tiling techniques to identify MDHAR6 mutants. In the absence of a working copy of MDHAR6, plants are able to detoxify TNT. We have not yet studied the longer-term fate of the detoxified TNT, we do not think it is fully broken down, more likely that it is converted to less-toxic compounds which become bound-up in the plant biomass (incorporated into woody tissues).