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.