Defense mechanisms of alfalfa against cyclic tetramethylene tetranitramine (HMX) stress

Much attention has been paid to the environmental toxicity and ecological risk caused by cyclic tetramethylene tetranitramine (HMX) pollution in military activity sites. In this study, the response mechanism of alfalfa plants to HMX was analyzed from the aspects of the photosynthetic system, micromorphology, antioxidant enzyme system, mineral metabolism, and secondary metabolism, in order to improve the efficiency of plant restoration. Exposure to 5 mg·L-1 HMX resulted in a significant increase in leaf N content and a significant increase and drift of the Fourier transform infrared protein peak area. Transmission electron microscopy images revealed damage to the root system subcellular morphology, but the plant leaves effectively resisted HMX pressure, and the photosynthetic parameters essentially maintained steady-state levels. The root proline content decreased significantly by 23.1-47.2 %, and the root reactive oxygen species content increased significantly by 1.66-1.80 fold. The roots regulate the transport/absorption of many elements that impart stress resistance, and Cu, Mn, and Na uptake is significantly associated with secondary metabolism. The metabolism of roots was upregulated in general by HMX exposure, with the main differences appearing in the content of lipids and lipid-like molecules, further confirming damage to the root biofilm structure. HMX causes an imbalance in the energy supply from oxidative phosphorylation in roots and generates important biomarkers in the form of pyrophosphate and dihydrogen phosphate. Interestingly, HMX had no significant effect on basic metabolic networks (i.e., glycolysis/gluconeogenesis and the tricarboxylic acid cycle), confirming that alfalfa has good stress resistance. Alfalfa plants apparently regulate multiple network systems to resist/overcome HMX toxicity. These findings provide a scientific basis for improving plant stress tolerance and understanding the HMX toxicity mechanism.