Uptake and metabolism of four isomers of dinitrotoluene in A. thaliana and their sequestration in feeding aphids: a phytoremediation perspective

Abstract

Plants, being sessile, have wide variety of metabolic pathways to detoxify hazardous compounds therefore ensuring the success of phytoremediation of a wide variety of xenobiotics. One such a compound is dinitrotoluene (DNT) that is available as a mixture of six different isomers. Phytoremediation of DNTs is a cost effective, eco friendly and preferable approach compared to commercially available chemical and physical remediation systems. DNTs are toxic to plants at higher concentrations, however lower concentrations are tolerable. The present study considers the uptake of DNTs in the model plant Arabidopsis thaliana to observe the potential of phytoremediation and considers the metabolites during different phases of detoxification. Further it evaluates the possible propagation of DNT and its metabolites in ecosystem through a model system of plant-aphid interaction. The first step in the study included establishment of the toxicity of four different available isomers of DNT (2,6-DNT, 2,3-DNT, 3,4-DNT and 2,4-DNT) in A. thaliana. This was achieved by root growth analysis in Phytagel (axenic) and hydroponic media (non axenic), root and shoot ratio in Phytagel and hydroponic media and finally a time course reduction in root length in hydroponic media. The results obtained from the study showed that all isomers of DNT have different toxicities (EC50 for 2,6-DNT, 2,4-DNT, 3,4-DNT and 2,3-DNT was 13.3 mg /L ,0.33 mg /L 0.23 mg /L and 0.13 mg /L of Phytagel media). At lower concentrations these DNTs were tolerable but at higher concentration they induced biomass inhibition. The uptake and metabolism of different DNT isomers was analysed by GC-MS in A. thaliana cultivated in Phytagel and hydroponic media amended with different concentrations of DNT isomers. The analysis of metabolites showed that the major metabolites of DNT were aminonitrotoluenes (ANTs), the concentration of which varied with the concentration of DNTs in the media (ranged from 0.54-5.2 μg /g dry tissue wt. of A. thaliana for 2A6NT; 20.03 to 280.3 ng /g dry tissue wt. of A. thaliana for 2A4NT and 11.34 to 508.39 ng /g dry tissue wt. of A. thaliana for 4A2NT; 32.8 to 675.22 ng/g dry tissue wt. of A. thaliana for 2A3NT; 41.23 to 564.32 ng /g dry tissue wt. of A. thaliana for 3A4NT). The qualitative and quantitative analysis of possible DNT based conjugates was carried out by LC-MS/MS analysis for A. thaliana cultivated in liquid MS media (axenic). The quantitative analysis of DNT based conjugates indicated ten significant conjugates with higher concentrations of O- and C- glucosides, mostly with hydroxylaminonitrotoluene. The abundant metabolites of DNT (aminonitrotoluene) have been observed to have lesser conjugation. The concentration of DNT based conjugates were observed to be indirectly related to the concentration of metabolites (increase with the decrease in the concentration of metabolites) and their concentrations were 102 times higher than aminonitrotoluenes. The concentration of conjugates varied with the type of isomer in the media. The effect of DNT treatment to herbivore feeders was evaluated through analysis of survival, fecundity and growth of Brevicoryne brassicae (specialist) and Myzus persicae (generalist) aphids on DNT treated A. thaliana at different concentrations. The DNT based metabolites were evaluated in the feeding aphid species to observe the possible propagation. Finally, the aphids were observed to tolerate lower concentrations of DNT treatments and their survival, fecundity and growth was highly compromised with high concentrations of DNT in the medium with B. brassicae showed higher sensitivity than M. persicae (in M. persicae LC50 for 2,6-DNT, 2,4-DNT, 3,4-DNT and 2,3-DNT was 13.9 mg /L , 0.34 mg /L , 0.138 mg /L and 0.14 mg /L of Phytagel media and in B. brassicae LC50 for 2,6-DNT , 2,4-DNT, 3,4-DNT and 2,3-DNT was 11.48 mg /L , 0.24 mg /L , 0.1 mg /L and 0.08 mg /L of Phytagel media). Both aminonitrotoluenes and glucosyl conjugates were present in both aphid species and their concentrations were directly related with the concentration of metabolites in the plants on which they were reared. GC-MS analysis showed that B. brassicae managed to accumulate more metabolites than M. persicae. LC-MS analysis showed that the type of conjugates varied with the type of isomer. Therefore this study shows that if Phytoremediation of DNTs is considered then plants convert the toxic DNTs to their polar (less toxic) reduced metabolites and glucosides which could potentially be transported to the feeding aphids.