In our last post on this paper, we introduced the concept of using plants for TNT bioremediation, why TNT is a pollutant to be bioremediated, and the logic behind a study that wants to know why plants aren’t efficient at it yet. Here, we are going to explore the science a little bit. How did the scientists confirm that an MDHAR6 mutation would give plants resistance to TNT and enable them to grow in faster rates? And what exactly is the mechanism by which MDHAR6 makes TNT toxic to wildtype (WT) plants, slowing their growth?
What did they do in lab to answer their questions?
We know that a frameshift mutation in MDHAR6 restores Arabidopsis growth rates under TNT pollution. The question we know want to answer is – how?
In Fig. 3, we are presented with the results of experiments that seek to answer just that question. Fig. 3A shows the percentage of MDHAR6 activity in leaf and root tissue of all three MDHAR6 mutants; they are quantified as a percentage of the enzyme activity in the WT. Thus, in the favored MDHAR6-1 mutant, leaf MDHAR6 enzyme activity is only ~80% of that in the WT when grown in clean soil. Add the TNT, and enzyme activity drops to ~60% of the activity in the WT. In root tissues, this drop in enzymatic activity is even greater. (For my mathematically inclined readers, the asterisks denote where statistically significant differences between mutant and WT enzyme activity occur, as confirmed by a student’s t-test.)
The next few graphs in Fig. 3 depict EPR spectra – both simulated and experimental. EPR (Electron Paramagnetic Resonance) is essentially NMR (Nuclear Magnetic Resonance; a technique you will learn in your organic chemistry class), but for molecules with unpaired electrons. It is a tool that allows you to identify molecules by their spectra patterns.
So in Fig. 3B-D, we have both simulated – theoretical – EPR spectra for various molecules, coupled with their experimental data. Now remember, EPR is good at detected molecules with unpaired electrons – it can detect superoxides. TNT is a non-radical molecule, so its spectrum would be flat – nothing detected. Thus, when we detect the presence of radicals in the MDHAR6 + TNT mixtures, we know some sort of reaction is occurring to alter the TNT.
The DMPO, DEPMPO, and DMPO-OH simulated data are there for reference: these are molecules with unpaired electrons. There are also three flat graphs, depicting various controls: there’s a flat read of MDHAR6 without TNT, for example. Nothing radical expected there. When MDHAR6 is denatured and mixed with TNT, the enzyme is deactivated and can’t react with it, resulting in another flat read.
Finally, the scientists mixed MDHAR6 + TNT + SOD, resulting in a flat read/no radical detected. What is SOD? It’s superoxide dismutase – an enzyme that resolves the unpaired electron in a radical, stabilizing it as oxygen or hydrogen peroxide. It deactivates the mechanism by which MDHAR6 reacts with TNT.
This chemical data strongly suggests the reason why an MDHAR6 mutation protects plants from TNT: an unmutated, activate MDHAR6 enzyme reacts with TNT to produce free radicals, sickening the plant. We know that plants grown under superoxide exposure will (when stained with a dye) develop a dark, sickly color (the dye DAB darkens when oxidized). Fig. 3E hits the nail on the head with just this observation: WT plant roots grown with TNT and stained with DAB darken and shrivel; plants with the MDHAR6 mutation have roots that look nice and healthy.
Thus, this data corroborates the suggested reaction shown in Fig. 4: MDHAR6 reacts with TNT to produce a nitro-radical version of TNT, resulting in oxidative stress in the mitochondria. This, then, is the means by which TNT sickens plants. By deactivating the enzyme with a mutation, we effectively immunize the plants against TNT.
Exploring herbicidal applications of this discovery, the scientists wrap up by looking at the effects of MDHAR6 mutations on plant response to other chemicals containing nitro-groups (and thus, chemicals that can react with WT MDHAR6; this data is summarized in Fig. 5.)
This study used genetics and chemistry to solve an environmental problem: how can we develop a plant strain effective in the bioremediation of a particularly insidious pollutant? After identifying a genetic mutation that protected plants from TNT toxicity, they were able to discover the chemical mechanism of toxicity in WT plants. This research, then, us provide us with a new set of chemicals & genetic manipulations that can be utilized for herbicide development for agricultural applications, as well as advance us in the bioremediation of TNT.
With a single frameshift mutation in the plant Arabidopsis, you can restore plant growth rates under TNT pollution conditions, enabling them to accumulate biomass at a normal rate, take up TNT, and then – detoxify it, via innate biosynthetic pathways already present in the organism.
Johnston, E. J., Rylott, E. L., Beynon, E., Lorenz, A., Chechik, V., & Bruce, N. C. (2015). Monodehydroascorbate reductase mediates TNT toxicity in plants. Science, 349(6252), 1072–1075. https://doi.org/10.1126/science.aab3472