3.4. SA, Ca2+ and NO recover Ni2+-induced damage in nitrogen metabolism status
Nitrogen metabolism is an important event which directly affects growth of the organisms. The inorganic nitrogen contents and related enzyme activities were differentially affected by Ni2+, SA, Ca2+, NO, and c-PTIO or/ and EGTA. Upon Ni2+ exposure, Anabaena cells exhibited a decrease upto 34 and 29% in NO3⁻ and NO2⁻ uptake rate, respectively; however these were recovered after SA, CaCl2 and SNP supplementation. Interestingly, this recovery in NO3⁻ and NO2⁻ uptake rate was further arrested when c-PTIO or/ and EGTA were added in culture medium (Fig. 3A,B). This inhibition in NO3⁻ and NO2⁻ uptake rate could be due to: (i) alteration in cellular homeostasis and membrane permeability as increased ROS levels, lipid peroxidation and electrolyte leakage was observed in the present study (Fig. 4), and (ii) impaired photosynthetic ETC decreased the photosynthetic rate (Fig. 2) and consequently the ATP pool, which is used as energy source by ABC-type transporter proteins that are essential for facilitating the entry of NO3⁻ and NO2⁻ in cyanobacterial cells (Flores and Herrero, 1994); therefore, Ni induced reduction in NO3⁻ and NO2⁻ uptake rate could be credited to decreased ATP pool, which is an outcome of impaired ETC as advocated by Rai et al. (1995) in Cu and UV-B stressed Anabaena doliolum . The two important enzyme of N-metabolism i.e. NR and NiR by successive reactions convert NO3⁻ into NO2⁻ and then NO2⁻ into highly toxic NH4+, respectively. Results revealed that both NR and NiR enzyme activities were sharply decreased upon Ni2+ exposure which was aggravated when Ni+SA treated cells were supplemented with c-PTIO or c-PTIO+EGTA (Fig. 3C,D). Ohmori and Hattori (1970) have proposed that NO3⁻induces NR activity while NO2⁻ induces NiR activity; therefore decline in NR and NiR activities could be directly interrelated with decreased NO3 and NO2⁻ uptake rate, (Fig. 3A,B). Another possibility behind this could be that NR needed sulfhydryl (-SH) groups for its catalytic action as suggested by Sharma and Dubey (2005); therefore Ni2+ by binding with -SH groups of the active sites of the enzyme might have altered the NR activity (Fig. 3C). Besides this, the limited availability of NO2⁻ ions which mainly arises from NR-catalyzed NO3⁻ reduction, might have decreased the NiR activity (Fig. 3D). Further, upon SA, CaCl2 and SNP application to the culture medium, a significant alleviation in Ni2+-induced decrease in enzyme activities of cyanobacterial cells was noticed thereby suggesting the improvement in the NO3⁻ and NO2⁻ uptake rate; while the sharp decrease in enzyme activities upon c-PTIO or c-PTIO+EGTA supplementation to Ni+SA treated cells suggest that in absence of Ca2+ and/ or NO, SA was unable to cope up with the Ni2+-induced toxic effect. Due to reduced NR and NiR activities, toxic NH4+accumulate in the cells, which rapidly is assimilated into organic compounds by the activity of glutamine synthetase (GS) enzyme, a primary route of NH4+ assimilation in cyanobacteria. Ni2+-stress significantly declined the GS activity by 33% in respect to control value (Fig. 3E), which could be due to the oxidative modifications of the enzyme. The GS along with GOGAT enzyme produces glutamate to synthesize numerous amino acids; therefore, decreased GS and GOGAT activities under Ni2+, Ni+SA+Ca+c-PTIO and Ni+SA+c-PTIO+EGTA, may carbon/ nitrogen homeostasis and consequently the growth of the cyanobacteria (Fig. 1A). Our results are in corroboration with the findings of Rai et al. (1998). The GS and GOGAT activity needed photosynthesis products ATP and NADPH (Muro-Pastor et al., 2003), for its functioning; therefore, decreased photosynthetic rate (Fig. 2A) might have decreased the ATP pool and NADPH and hence the GS activity. However, Ni2+-induced reduction in GS and GOGAT activity was appreciably alleviated by SA, CaCl2 and SNP treatment (Fig. 3E,F) indicating the betterment in NH4+-assimilation and its incorporation into glutamate compared to Ni2+ as well as Ca2+ and NO scavenger treated cells. In contrast to GS and GOGAT, aminating activity of GDH was sharply increased under Ni2+-stress (62%) as well as Ni+SA+c-PTIO+EGTA (68%) treated cells in respect to control (Fig. 3G), which might have occurred to improve the NH4+-assimilation, that was inhibited due to decreased GS and GOGAT activity. Indeed Skopelitis et al. (2006) have also reported that during stress when GS/GOGAT cycle for NH4+-assimilation doesn’t operate efficiently, then GDH activity increases to relieve the pressure of NH4+-accumulation and also to provide the glutamate for the biosynthesis of Pro like protective compounds. On the other hand, SA, CaCl2 and SNP addition to Ni2+-stressed culture medium showed decrease in GDH activity while improvement in the activities of GS/GOGAT enzymes, which may be due to decreased NH4+ content that shifted the cycle towards GS/GOGAT pathway.