Nitric oxide and ascorbic acid confer cadmium (Cd) tolerance by improving plant terpenoid metabolism and epigenetically modifying DNA methylation

This study investigated the efficacy of incorporating nitric oxide (NO; 10 μM) and ascorbic acid (Asc; 10 μM) into the culture medium to confer cadmium (Cd; 5 μM) tolerance in thyme (Zataria multiflora). The phytotoxicity of Cd resulted in a decrease in shoot biomass, which NO or Asc mitigated. Adding Asc and NO to the culture medium was associated with substantial DNA hypomethylation. The NO + Cd and Asc + Cd treatments were accompanied by an increase in the unmethylation percentages, about 3-fold higher than the control. The hemi-methylation percentages in the Asc-supplemented seedlings also displayed an upward trend. The transcriptional upregulation in the γ-terpinene synthase (TPS) gene resulted from the applied elicitors, especially NO. In response to the NO and Asc treatments, the transcription of two cytochrome P450 monooxygenase genes (CYP71D178 and CYP71D180) went up. Incorporating Asc or NO into the culture medium enhanced the concentrations of proline, carvacrol, and thymol metabolites. Employing NO or Asc mitigated the 43% decrease in protein content due to the Cd cytotoxicity. The NO and Asc applications improved the activity of the phenylalanine ammonia-lyase (PAL) enzyme. NO and Asc utilization increased the accumulation of flavonoids. NO and Asc also up-regulated the activities of two enzymatic antioxidants (catalase and peroxidase). Collectively, this study provided novel insight into how Asc or NO confers Cd tolerance by epigenetically remodeling DNA methylation, transcriptionally up-regulating terpenoid and phenylpropanoid metabolism, increasing proline concentration, and improving antioxidants.