A Mechanistic Study on the Toxic Effect of Copper Oxide Nanoparticles in Soybean (Glycine max L.) Root Development and Lignification of Root Cells

Copper oxide nanoparticles (CuONPs) are widely used in several products and their release into the environment can cause toxicity to major food crops. In this study, toxic responses as a result of CuONPs exposure were studied in soybean (Glycine max L.) seedlings. The plants were grown in 1/2 strength Murashige and Skoog medium containing 0, 50, 100, 200, 400, and 500 mg/L of CuONPs in a growth chamber at 26 +/- 2 A degrees C with 16/8 h light/dark photoperiod for 14 days. The toxic effects of CuONPs were tested on the shoot and root development, total chlorophyll content, hydrogen peroxide generation, peroxidase (POD) enzyme activity, and lignification of root cells. The mRNA expression of different genes involved in lignin biosynthesis viz. phenylalanine ammonia lyase (PAL), cinnamate 4-hydroxylase (C4H), cinnamyl alcohol dehydrogenase (CAD), peroxidase 2 (POD2), peroxidase 4 (POD4), and peroxidase 7 (POD7) was studied using real-time polymerase chain reaction. Exposure to 500 mg/L of CuONPs significantly reduced the shoot growth, weight, and total chlorophyll content. However, the root length and fresh weights were significantly reduced at all concentrations of CuONPs exposure. Exposure to 100, 200, 400, and 500 mg/L of CuONPs significantly increased the hydrogen peroxide level, POD activity, and lignin contents of roots. Treatment with 2,7-dichlorofluorescein diacetate indicated a concentration-dependent increase in reactive oxygen species generation in roots. Staining with phloroglucinol-HCl revealed a concentration dependant increase in lignification of root cells. The expression levels of PAL, C4H, and CAD genes were significantly up-regulated upon exposure to 100, 200, and 400 mg/L of CuONPs. Significant up-regulation in the expression levels of POD2 and POD4 genes was observed upon exposure to 100, 200, 400, and 500 mg/L of CuONPs. Exposure to 200, 400, and 500 mg/L of CuONPs resulted in significant up-regulation of POD7 gene. These results for the first time show that exposure to CuONPs causes enhanced lignification of root cells and thereby affect root development in soybean seedlings.