The analysis of variance showed significant differences among the treatments for all the characters under study (Table 1). The total dry weight of the seedling decreased under both the metal treatments, except for Cu 50µM (Table 2) where the seedling dry weight remained at par with the unstressed control. However, the extent of decrease was more as the molar concentration of the metals increased in the growing medium. Such decrease can be attributed to the decrease in number of cells and reduction in the elongation rate of developing root and shoot. However, lead, in general, was found to be more detrimental for seedling growth in comparison to copper as it was also reported earlier by
Ilic et al., (2015) and
Monalisa et al., (2017). In the present study, the highest intensity (200 µM) of copper and lead stress led to a reduction of seedling dry weight by 32.50% and 52.50%, respectively over that of unstressed control.
Heavy metals at higher concentration decrease the leaf chlorophyll content
(Thapar et al., 2008). Copper-induced chlorosis may result from the inhibition of biosynthesis and damage in chlorophyll structure
(Kupper et al., 2003). In general, the total chlorophyll content of leaf in the present experiment decreased (Table 2) under both the metal treatments, except for copper at 50 µM where, it recorded 5.60% increase over control. Copper, being an essential element for plant, might attribute for higher rate of pigment biosynthesis at lower concentration. In contrast, lead caused reduction in leaf chlorophyll with the effect being more drastic as compared to corresponding concentrations of copper. This decrease in pigment content under Pb stress, might lead to subsequent reduction in photosynthetic capacity
(Zhou et al., 2018). The highest intensity of copper and lead stress led to a reduction in total chlorophyll content by 22.93% and 24.17%, respectively, over that of unstressed control.
Proline, under abiotic stress acts as a compatible solute, osmo-protectant, free radical scavenger, as well as antioxidant
(Zhang et al., 2008; Hayat et al., 2012). However, different abiotic stresses may have varying effects on leaf proline depending upon the intensity of stress. In the present experiment, the leaf proline content decreased under moderate to high concentrations of both the metals with the effects being more as the molar concentration increased (Table 2). On the contrary, mild metal stress (50 µM) induced increase in leaf proline over that of control indicating an attempt of the plant to combat dehydration shock. However, lead, at its highest concentration, was found to be more detrimental for proline content in comparison with copper for this variety of ricebean, a finding corroborating the early observation of
(Ilic et al., 2015). The leaves recorded 16.51% and 27.36% decrease in proline content at an equimolar concentration (200 µM) of copper and lead stress, respectively, over that of control.
The relative leaf water content (RLWC) is one of the reliable parameters to know the water status in plants. The RLWC in the present experiment also decreased under both the metal treatments with the effects being more as the molar concentration increased (Table 2). The observed decrease in RLWC under metal stress might be a consequence of impairment of root growth resulting from mitotic disturbance. The RLWC registered more adverse effects of copper stress in comparison with lead stress at the highest concentration, although a reverse trend was noted at the lower levels. Excessive concentration of copper generally causes low biomass accumulation, chlorosis, inhibition of growth and photosynthesis, altered water balance and nutrient assimilation and senescence, which ultimately lead to death (
Ozdener and Kutbay 2009;
Ling and Jun 2010).
The extent of leaf membrane damage was measured by determining the level of lipid peroxidation which in turn, was estimated as the content of thiobarbituric acid reactive substances (TBARS). The increased values (Table 2) of TBARS under copper and lead toxicity indicated enhanced generation of free radicals under metal toxicity in the present experiment. It might be further noted that the lead stress was found to be more damaging for leaf membrane than copper stress at all concentrations. The increased level of TBARS indicated the enhanced production and accumulation of reactive oxygen species (ROS) due to Pb toxicity
(Thakur et al., 2017; Vasavi et al., 2012). The variety Bidhan-1 recorded a 53.48% and 77.54% increase in lipid peroxidation over control at the highest concentration of copper and lead respectively.
For protection against ROS, plant cells contain both enzymatic and non-enzymatic components. Out of the enzymatic components, superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX) and guaiacol peroxidase (GPOX) are found to be very important
(Atta et al., 2022). The leaf APX activity showed the same pattern of change in copper and lead stress, a remarkable enhancement over control under mild stress followed by a decreasing trend as the intensity of stress increased (Table 3). The higher doses of lead caused decrease in the activity of this enzyme over that of control. High amount of copper Cu caused oxidative damage and induced anti-oxidative defence by enhancing activities of ascorbate peroxidase
(Ali et al., 2006).) However, lead, in general, was found to be more detrimental for APX activity in comparison with copper for this variety of ricebean as was also observed in other crops
(Singh et al., 2011, Ilic et al., 2015).
Like APX, the GPOX activity also revealed the same trend (Table 3) under both the metal treatments where the mild and medium stress-induced much higher activity which then decreased somewhat as the stress increased. Earlier
Wang et al., (2004) reported alleviation of oxidative damage under copper stress by enzymatic system involving GPOX, SOD, CAT and APX. Whereas lower induction of GPOX under lead stress as compared to copper was noted earlier by
Singh et al., (2011) and
Ilic et al., (2015). In the present experiment, the variety Bidhan 1 recorded 18.56% increase in the content of leaf GPOX activity under copper toxicity at 200 ìM concentration and there was almost no change under lead stress as compared to that of control.
The leaf SOD activity increased significantly under both the metal treatments at all concentrations except for Pb 200 μM which registered 24.67% decrease over control (Table 3). From the present study, it was found that SOD recorded much higher activity under all concentrations of Cu than Pb. This was expected for copper as this metal, by itself, is a cofactor of SOD. In case of Pb stress a significant increase in the SOD activity than control was seen for low and medium concentration but at the highest concentration of Pb it decreased. The observed increase in SOD activity under lead stress might be a consequence of upregulation via an increase in levels of O
2-
(Chongpraditnum et al., 1992).
The leaf CAT activity registered a sigmoidal pattern of change under varying concentrations of Cu, an increase over control under 50 μM, reaching the peak at 100 mM followed by a decline (3.87%) at the highest concentration of Cu (Table 3). In contrast, the enzyme showed a decreasing trend concomitant with a rise in the concentration of lead with the highest concentration registering 9.68% decrease in enzyme activity. However, such decrease in the activity of antioxidative enzymes along with the rise in heavy metal concentration might be caused by either the direct action of ROS on the enzymatic proteins or on the inhibition of protein synthesis
(Mazhoudi et al., 1997).
