3.5. SA, Ca2+ and NO recover antioxidant system to neutralize Ni2+-induced oxidative stress
To define the interaction between SA, Ca2+ and NO in alleviating the detrimental effect of Ni2+ inAnabaena cells, the oxidative stress status of cyanobacteria was investigated. In the present study, excessive Ni2+accumulation revealed severe oxidative stress as evident by increased ROS (O2•─ and H2O2: 33 and 37%, respectively) levels, which consequently lead to oxidative injuries credited by lipid peroxidation and membrane damage as increased levels of MDA equivalents and electrolyte leakage were observed in the present study (Fig. 4) and hence decreased the growth of cyanobacteria. The possibility behind this could be the blockage in ETC of PSII, due to which most of the electrons slipped out and reacts with available molecular oxygen to produce O2•─. Prasad et al. (2005) and Jahan et al. (2020) have also reported the increased levels of ROS, MDA formation and electrolyte leakage in Ni2+ or/ UV-B stressed Glycine max L. and tomato seedlings, respectively due to higher Ni2+ accumulation. Further, SA, Ca2+ and NO addition to the culture medium counteracted the Ni2+-induced loss in cell structure and function by decreasing ROS levels and the indices of damage as manifested by decreased Ni2+ accumulation and increased NO content (Fig. 1) as was also reported by Tiwari et al. (2019a) and Singh et al. (2020a,b) in aluminium and As-stressedAnabaena and mustard seedlings, respectively. The increased SA, Ca2+ and NO might be involved in inducing several ROS-scavenging enzyme activities such as SOD, CAT, POD and GST (Fig. 5) as was discussed by Tiwari et al. (2019a,b) or increased NO content might have been involved in the formation of less toxic peroxynitrite (ONOOˉ) as was discussed by Peto (2011) in Cu-stressed auxin supplemented Arabidopsis seedlings. Interestingly, c-PTIO or/ and EGTA application to Ni+SA stressed cyanobacteria arrested the Ca2+ and NO induced effect in Ni2+-stressed cells, which was further confirmed by histochemical visualization by staining SOR, H2O2, lipid peroxidation and injury of plasma membrane integrity (more dense color under Ni2+and c-PTIO or/ and EGTA supplemented cells; however, upon SA, CaCl2 and SNP supplementation to Ni2+-stressed cells showed comparatively less intense color) (Fig. 4). While working on chromium-stressed Nostoc muscorum , Tiwari et al. (2018) have reported that kinetin recovered the growth of Cr-stressed cyanobacteria by up-regulating the activities of enzymatic antioxidants. Indeed in the present study, an increasing trend in the activities of enzymatic antioxidants: SOD, POD, CAT and GST was noticed that could have helped to overcome the damaging effect of Ni2+, and the activity of these enzymes was further increased upon SA, CaCl2 and SNP addition but this increment in the enzyme activities was arrested after c-PTIO or/ and EGTA treatment, most importantly under Ni+SA+Ca+c-PTIO and Ni+SA+c-PTIO+EGTA, and it was even less than that of control (Fig. 5). The SOD, POD and CAT act first fence against stress, which dismutate O2•─ into H2O2 and consecutively into H2O; therefore, increment in these enzyme activities might have speeded the reduction in ROS accumulation, but it was inadequate to overcome the huge Ni2+-induced c-PTIO and EGTA mediated ROS accumulation; thus still high levels of ROS were noticed (Fig. 4), especially under c-PTIO or/ and EGTA. Similarly, glutathione-S -transferases (GST) enzyme regulates Ni2+-toxicity by eliminating xenobiotics through conjugation with GSH; therefore, increase in GST activity (Fig. 5D) might have reduced the availability of Ni2+ through its conjugation, hence might helped the cyanobacteria cells to overcome the stress situation. These findings suggest the regulatory role of Ca2+ and NO in SA induced signaling. Our results are in accordance with the earlier findings of Tiwari et al. (2018; 2019a,b) and Singh et al. (2018a, 2020a) in Nostoc muscorum and mustard seedlings, respectively.