Seeds were sown in earthen pots

Seeds were sown in earthen pots (25×25cm) filled with 5kg of soil containing sandy loam soil and farmyard manure (6:1v/v), urea, single superphosphate and muriate of potash were added at 40, 138 and 26mgkg−1 of soil, respectively. Soil in the selected pots was mixed with Cd (0, 25, 50 or 100mg CdCl2kg−1 of soil) and watered on alternate days. Both the varieties were sampled at two growth stages (30 and 60 DAS). The plants were removed from the pots along with the soil and were dipped in a bucket filled with tap water. The plants were gently moved to remove the adhering soil particles.

Results

Discussion
Plant genotypes differ in their ability to take up and translocate soil-amended Cd from roots to shoots (Metwally et al., 2005). The ability to check root uptake and aerial distribution of Cd depends on its binding to extracellular matrix, root efflux, intracellular detoxification and its transport efficiency (Marchiol et al., 1996; Akhtar and Macfie, 2012; Meng et al., 2012). In this study Varuna, compared with RH-30, accumulated lesser quantity of Cd both in root and shoot tissues (Fig. 2F and G) because of the reasons mentioned earlier. The absorbed Cd accumulates preferably in plant roots followed by shoots, which often restricts the uptake and distribution of other nutrients (Gomes et al., 2013 and Fig. 2F and G). This study indicates, the level of the metal increased with a progressive increase in the soil Cd content (0, 25, 50 or 100mgkg−1) both in root and shoot.
Cadmium uptake at toxic level causes mineral deficiency, desiccation and cellular metabolic disturbances (Marshner, 2012; Gomes et al., 2013) in plants. Cadmium alters the membrane permeability and hence cellular LWP (Fig. 2D). Cd affected membrane potential and proton pump activity could restrict the growth of maize plants (Karcz and Kurtyka, 2007). Moreover, Cd brought about aquaporin mediated reduction in maize root hydraulic conductivity that reduced the cellular turgor and leaf gli1 even without changing transpiration (Ehlert et al., 2009). Therefore, an increase in Cd concentration both in root and shoot (Fig. 2F and G) partially damaged the membrane which resulted in decreased LWP (Fig. 2D). However, proline accumulation is an adaptive mechanism to counter osmotic stress caused by decreased LWP; therefore, it reestablishes the LWP and augments the loss of cellular osmoticum (Alia and Saradhi, 1991; Albert et al., 2012). Varuna accumulated more proline than RH-30 which could have favored the maintenance of LWP in this cultivar (Fig. 2D and E).
The Cd binding to root epidermal membrane affects the functioning of transporter proteins either through direct binding to the ion transporters or via membrane assisted ROS production. The competitive exclusion of the substrate () potentially impeded the NR activity (Hernandez et al., 1996; Campbell; 1999; Fig. 1H) or, alternatively, the metal could have bound with the –SH group, directly affecting the enzyme structure and its functions (Choudhary and Singh, 2000). Cadmium induced supra-optimal generation of ROS could interfere with the active state of NR rendering it inactive. The NR activity exhibited a progressive decline in response to increasing dose of Cd (Fig. 1H). However, proline protects membranes and subcellular structures, hydrates the enzymes to restore their activity and neutralizes reactive oxygen/nitrogen species (Hare and Cress, 1997; Kavi Kishor et al., 2005), its increased detoxification capacity (Fig. 2A–C) may have potentially protected the NR activity more effectively in Varuna as compared to RH-30.
Besides proline, antioxidant enzymes are also the key players in maintaining cellular redox status and stress induced plant tolerance (Kavi Kishor et al., 2005; Gill and Tuteja, 2010; Gill et al., 2011; Hayat et al., 2012). The higher activity of antioxidant enzymes (i.e. POX, CAT and SOD) was in proportion to the progressive increase in the concentration of Cd (CdCl2; 0, 25, 50 or 100mgKg−1 of soil; Fig. 2A–C). Moreover, the per cent increase in antioxidant enzymes was more in Varuna as compared to RH-30 (Figs. 2A–C). Mohamed et al. (2012) has shown in B. juncea that the higher activity of antioxidative enzymes offers a greater detoxification efficiency which provides better resistance to a plant variety against heavy metal induced oxidative stress.