3.1. SA, Ca2+ and NO recover Ni2+-induced damage in growth
To examine the SA induced Ca2+ and NO signaling in easing Ni2+‒induced toxicity, Anabaena PCC 7120 cells were exposed to different donors, scavengers and inhibitors of SA, Ca2+ and NO. Nickel at its tested dose significantly declined the growth (dry weight) of Anabaena by 31%, which is attributed to: (i) higher Ni2+ accumulation in the cell (Fig. 1D), (ii) reduction in photosynthetic pigments content (Table 1) and PSII performance (Fig. 2C), which subsequently decreased photosynthetic rate (Fig. 2A), (iii) overproduction of ROS (Fig. 4), (iv) reduction in NO3 and NO2 uptake rate (Fig. 3A,B), and (v) decrease in EPS content (Fig. 1B). However, under similar condition, addition of SA, CaCl2 and SNP in the growing medium counteracted Ni2+‒induced negative impact on DW (12, 11 and 7% respectively) of test cyanobacteria, which is in agreement with the higher NO accumulation than in the Ni2+- stressed cyanobacterial cells alone (Fig. 1C). The SA, Ca2+ and NO induced progressive response on growth have also been reported by Tiwari et al. (2018, 2019a) and Singh et al. (2020a,b) in Anabaena PCC 7120 and mustard, respectively. Further, in order to study the possible link between SA, Ca2+ and NO; when Ni+SA+Ca and Ni+SA+NO treated cyanobacteria cells were supplemented with c-PTIO (a NO scavenger) and EGTA (a Ca2+ scavenger), respectively; a significant reduction in DW was observed, thereby satisfying the fact of involvement of Ca2+ and NO in signaling event. Interestingly, the growth of c-PTIO supplemented Ni+SA+Ca treated cyanobacterial cells was comparatively more affected even in presence of Ca2+(Fig. 1A), suggesting that NO is required for the maximal and constant Ca2+ signaling, which corresponds to reduce NO content in presence of c-PTIO (Fig. 1C). The report of Xu et al. (2013) have strengthened our findings that by promoting NO release, ABA hormone protected tall fescue plant from oxidative stress, and Singh et al. (2020a,b) have suggested that NO is essential in Ca2+mediated signaling event in arsenic-stressed mustard seedlings.
Results for intracellular Ni2+ accumulation showed higher Ni2+ accumulation under Ni2+-stressed condition, which was neutralized upon SA, CaCl2 and SNP treatment to Ni2+-stressed cyanobacterial cells; however, in presence of c-PTIO and EGTA, even SA was unable to limiting Ni2+-accumulation; therefore, highest Ni2+-accumulation was found in Ni+SA+c-PTIO+EGTA even than that of Ni2+-stressed cyanobacterial cells alone (Fig. 1D).