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.