Cd induced generation of free radical species in Brassica juncea is regulated by supplementation of earthworms in the drilosphere.

The antioxidant defense system of Brassica juncea under Cd stress was examined on supplementation of earthworms in the rhizosphere at different concentrations of Cd (0.50, 0.75, 1.00 and 1.25 mM i.e. 56, 84, 112 and 140 mg kg respectively). Seedlings were raised in small pots containing soil spiked with Cd and earthworms under controlled conditions for 15 days. Improved Cd accumulation, as well as enhanced plant dry weight and metal tolerance were observed following the addition of earthworms. Earthworm supplementation reduced reactive oxygen species (ROS) generation by 7.3% for hydrogen peroxide (HO), 7.1% for superoxide anion (O), and 8.4% for malondialdehyde (MDA) in plants treated with 1.25 mM (140 mg kg) Cd. Confocal microscopy revealed improved cell viability and reduced HO content due to enhanced antioxidative activity. Activity and expression levels of genes coding for antioxidative enzymes (superoxide dismutase; SOD, catalase; CAT, guaicol peroxidase; POD, glutathione reductase; GR, and glutathione-S-transferase; GST) were higher in plants raised in soils inoculated with earthworms, with expression of SOD increasing by 58.8%, CAT by 75%, POD by 183%, GR by 106.6%, and GST by 11.8%. Moreover, plant pigment (chlorophyll a, chlorophyll b, total chlorophyll, and carotenoids) concentrations increased by 8%, 9.1%, 9.1%, and 7.7% respectively, in plants grown in soils supplemented with earthworms. The results of our study suggest that the addition of earthworms to soil increases antioxidative enzyme activities, gene expression in plants, and ROS inhibition, which enhances tolerance to Cd during the phytoextraction process.