Creation of Environmentally Friendly Super "Dinitrotoluene Scavenger" Plants

Abstract

Pervasive environmental contamination due to the uncontrolled dispersal of 2,4-dinitrotoluene (2,4-DNT) represents a substantial global health risk, demanding urgent intervention for the removal of this detrimental compound from affected sites and the promotion of ecological restoration. Conventional methodologies, however, are energy-intensive, susceptible to secondary pollution, and may inadvertently increase carbon emissions. In this study, a 2,4-DNT degradation module is designed, assembled, and validated in rice plants. Consequently, the modified rice plants acquire the ability to counteract the phytotoxicity of 2,4-DNT. The most significant finding of this study is that these modified rice plants can completely degrade 2,4-DNT into innocuous substances and subsequently introduce them into the tricarboxylic acid cycle. Further, research reveals that the modified rice plants enable the rapid phytoremediation of 2,4-DNT-contaminated soil. This innovative, eco-friendly phytoremediation approach for dinitrotoluene-contaminated soil and water demonstrates significant potential across diverse regions, substantially contributing to carbon neutrality and sustainable development objectives by repurposing carbon and energy from organic contaminants.

Keywords: 2,4-dinitrotoluene (2,4-DNT); carbon neutrality; complete degradation; rapid phytoremediation; sustainable development goals.

Figure 1

Creation of super “dinitrotoluene scavenger” plants. A) Gene cassette schematic representation of the complete 2,4‐DNT degradation, as designed in this study. dntAaS, dntAbS, dntAcS, and dntAdS (encoding four subunits of DNT dioxygenase); dntBS (encoding methylnitrocatechol monooxygenase); dntDS (encoding trihydroxytoluene oxygenase); dntGS (encoding bifunctional isomerase/hydrolase); dntES (encoding CoA‐dependent methylmalonate semialdehyde dehydrogenase). B) Real‐time polymerase chain reaction analysis of the expression of genes involved in the synthetic 2,4‐DNT degradative pathway. C) Western blot analysis of the expression of proteins involved in the synthetic 2,4‐DNT degradative pathway. DS_R1 and DS_R2 represent two modified rice plants; WT represents wild‐type rice plants.

Figure 2

2,4‐DNT resistance assay in the WT and dinitrotoluene scavenger rice seedlings. A) Representative images of the WT and dinitrotoluene scavenger rice seedlings grown in soil supplemented with 2,4‐DNT at concentrations ranging from 0 to 40 mg kg⁻¹. B) Comparison of the fresh weight of rice plants grown in medium supplemented with 0, 20, 30, and 40 mg kg⁻¹ 2,4‐DNT. C) Shoot length of rice plants grown in medium supplemented with 0, 20, 30, and 40 mg kg⁻¹ 2,4‐DNT. Data are presented as the mean ± SD (n = 6). NS, no significant difference. p‐values were calculated using the Student's t‐test. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Figure 3

Residual and accumulated 2,4‐DNT in the medium and plants. A) High‐performance liquid chromatography (HPLC) analysis of the residual 2,4‐DNT in medium‐cultivated wild‐type (WT) and dinitrotoluene scavenger rice plants. B) The fate of 2,4‐DNT in the medium‐cultivated WT and dinitrotoluene scavenger rice plants. C) HPLC analysis of accumulated 2,4‐DNT in the WT and dinitrotoluene scavenger rice plants. D) Comparison of 2,4‐DNT accumulation between the WT and dinitrotoluene scavenger rice plants. The WT and dinitrotoluene scavenger rice seedlings grown on medium containing 0, 20, 30, and 40 mg L⁻¹ 2,4‐DNT for 10 d. The asterisk at the top of the bar indicates different significances according to Student's t‐test. Error bars, mean ± SD (n = 2), *p < 0.05; **p < 0.01; ***p < 0.001.

Figure 4

Accumulation of 2,4‐DNT degradation intermediates in plants. A) Gas chromatography‐mass spectrometry (GC‐MS) analysis of the formation of 4M5NC. B) GC‐MS analysis of the formation of pyruvic acid. C) GC‐MS analysis of the formation of propionyl‐CoA. D–F) Relative concentration of 4M5NC, pyruvate, and propionyl‐CoA in the WT and dinitrotoluene scavenger rice plants. The asterisk at the top of the bar indicates different significances according to Student's t‐test. Error bars, mean ± SD (n = 2), *p < 0.05; **p < 0.01.

Figure 5

Mass isotopomer analysis of 2,4‐DNT degradation intermediates. A) Mass isotopomer analysis of citrate, succinate, and fumarate in DS_R1 line by GC‐MS. B) Calculation of mass isotopomer distribution of different succinate isotopologues by uniformization.

Figure 6

Schematics diagrams illustrating the mechanism of dinitrotoluene scavenger rice plants degrade 2,4‐DNT into an innoxious substance, upcycling of the 2,4‐DNT pollutant to a “green” feedstock and progress of rapid phytoremediation of 2,4‐DNT contamination. PDC, pyruvate dehydrogenase complex; PC, pyruvate carboxylase.

Figure 7

Phytoremediation of 2,4‐DNT‐contaminated soil. A) High‐performance liquid chromatography analysis of the residual 2,4‐DNT in soil cultivated with the WT and dinitrotoluene scavenger rice plants. B) The fate of 2,4‐DNT in soil cultivated with the WT and dinitrotoluene scavenger rice plants. The asterisk at the top of the bar indicates different significances according to Student's t‐test. Error bars, mean ± SD (n = 2), *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Figure 8

Bacterial community composition in soil cultivated with the WT and dinitrotoluene scavenger rice seedlings. A) The abundance percentages of the bacterial genera in the soil samples. B) The relative abundance of the genera among soil samples cultivated with the WT and DS_R1 rice seedlings. C) The relative abundance of the genera among soil samples cultivated with the WT and DS_R2 rice seedlings.