Copper-stress induced alterations in protein profile and antioxidant enzymes activities in the in vitro grown Withania somnifera L

Abstract

Withania somnifera L. seedlings were grown in half-strength MS (Murashige and Skoog) basal medium for 4 weeks and then transferred to full-strength MS liquid medium for 3 weeks. The sustainable plants were subcultured in the same medium but with different concentrations (0, 25, 50, 100 and 200 μM) of Cu for 7 and 14 days. The growth parameters (root length, shoot length, leaf length and total number of leaves per plant) showed a declining trend in the treated plants in a concentration dependant manner. Roots and leaves were analyzed for protein profiling and antioxidant enzymes [catalase (CAT, EC 1.11.1.6), superoxide dismutase (SOD, EC 1.15.1.1) and guaiacol peroxidase (GPX, EC 1.11.1.7)]. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of crude protein extracts showed the appearance of some new proteins due to Cu treatment. In plant samples grown with 25 and 50 μM of Cu, a rapid increase in antioxidant activities were noticed but at higher concentration (100 and 200 μM) the activities declined. Isoforms of CAT, SOD and GPX were separated using non-denaturing polyacrylamide gel electrophoresis and concentration specific new isoforms were noticed during the study. Isoforms of the antioxidant enzymes synthesized due to Cu stress may be used as biomarkers for other species grown under metal stress.

Keywords: Antioxidant enzymes; Copper stress; Protein profiling; SDS-PAGE; Withania somnifera L..

Fig. 1

Total soluble protein estimation under different Cu treatment (0, 25, 50, 100 and 200 μM) in leaf (a) and root (b) samples of W. somnifera L. The data plotted represent mean ± SE of replicates (n = 6). Symbol carrying different letters are significantly different at P ≤ 0.05 by Duncan’s multiple range test

Fig. 2

CAT activity (a, b) and CAT isoenzymes (c, d) detected in extracts of W. somnifera L. grown for a 7 days (a, c) and 14 days (b, d) period on liquid MS medium containing different concentrations of Cu (0, 25, 50, 100 and 200 μM). Statistical analysis was as in Fig. 1

Fig. 3

SOD activity (a, b) and SOD isoenzymes (c, d) detected in extracts of W. somnifera L. grown for a 7 days (a, c) and 14 days (b, d) period on liquid MS medium containing different concentrations of Cu (0, 25, 50, 100 and 200 μM). Statistical analysis was as in Fig. 1

Fig. 4

GPX activity (a, b) and GPX isoenzymes (c, d) activity detected in extracts of W. somnifera grown for a 7 days (a, c) and 14 days (b, d) period on liquid MS medium containing different concentrations of Cu (0, 25, 50, 100 and 200 μM). Statistical analysis was as in Fig. 1