3.3. SA, Ca2+ and NO revive
Ni2+-induced losses of photosynthetic pigments,
up-regulate photosynthetic rate and PSII photochemistry and
down-regulate respiratory rate
To further interpret the SA induced Ca2+ and NO
signaling on photosynthetic activity, photosynthetic pigments:
chlorophyll a (Chl a ), carotenoids (Car), and
phycobiliproteins [phycocyanin (PC), allophycocyanin (APC) and
phycoerythrin (PE)] were studied (Table 1). Nickel at its tested dose
caused a significant reduction in the photosynthetic pigments content
probably due to degradation of precursors or pigments’ biosynthesis
itself as argued by Prasad et al. (2005); that consecutively decreased
the photosynthetic capacity of tested cyanobacteria, which was in
conformity with the reduced DW of tested organism (Fig. 1A). SA,
CaCl2 and SNP treatment, on the other hand recovered the
Ni2+-induced damage in photosynthetic pigments content
which could partly attributed to improvement either: (i) in the gene
expression encoding protochlorophyllide oxidoreductase (Kusnetsov et
al., 1998; Verma et al., 2018), or (ii) in the phytol isomers, necessary
for pigment biosynthesis (Tiwari et al., 2019b). However, the
Ni2+-induced reduction in photosynthetic pigments
content was worsened when Ni+SA treated Anabaena cells were
supplemented with c-PTIO+EGTA, which decreased the photosynthetic rate
(Fig. 2A) and consequently the growth (Fig. 1A). The reduction in water
soluble PC pigment content under Ni2+-toxicity could
be its external location on thylakoid membrane and thus easily bleached
by ROS produced due to heavy metal (Tiwari et al., 2019a). However, SA,
CaCl2 and SNP treatment under similar condition
significantly declined the ROS levels, which indirectly improved the PC
content that ultimately favored photosynthetic activity than that of
stressed condition alone (Fig. 2A). Ni2+ significantly
reduced the photosynthetic rate which could be due to the reason that
after entering into cell this divalent cation depolarizes the membrane
by binding to it, thereby acidifying the cytoplasm and interferes with
photosynthesis and respiration both as suggested by Balaji et al. (2013)
in Spirulina strains.
Further, to deeply understand the SA induced Ca2+ and
NO signaling in structural and functional properties of PSII, the
biophysical traits deduced from OJIP transient curves were studied (Fig.
2C). Ni2+ exposure, strongly inhibited the oxygen
evolution rate by 31% in comparison to control of Anabaena cells
(Fig. 2A), indicate that photosynthetic apparatus especially the
oxygen-evolving complex (OEC) was the main target of
Ni2+, which could be due to interaction of
Ni2+ with 16 and 24 kDa extrinsic polypeptides causing
conformational changes and provoked the Ca2+ release
from OEC that ultimately inhibited electron transport activity as argued
by Boisvert et al. (2007) or might have inactivated the OEC (decreasedFm ). Further, upon c-PTIO or/ and EGTA
supplementation to Ni2+-stressed cyanobacteria cells,
the increment in Fo/Fv(efficiency of water splitting complex/ OEC) values indicate the
injurious effect on OEC, which could be due to obstruction in mineral
uptake (like Ca2+ and Mn2+)
necessary for OEC functioning (Najafpour et al., 2012), that ultimately
blocked electron transfer rate from donor side (OEC) to PSII (Kalaji et
al., 2014; 2016; 2017).
In the present study, a drastic change in JIP-test parameters under
Ni2+-toxicity suggests that PSII was the primary
target of Ni2+. The radar plot reveals that
Ni2+ addition in culture medium sharply increased the
initial fluorescence (Fo ) values, which could
results from (i) dissociation of LHCII from RC complexes, (ii) back flow
of electrons from plastoquinone (PQ) pool (Yamane et al., 2000), and
(iii) decrease in size and number of active photosynthetic reaction
centers (RCs) (Fv/Fo ); which
might have blocked the electron flow beyond QAˉ thus,
higher Fo was measured in presence of
Ca2+ and NO scavenger as was reported in
amoxicillin-stressed Synechocystis sp. (Pan et al., 2008). While
decrease in the variable fluorescence (Fv ) and
maximum fluorescence (Fm ) values could be due to
(i) decrease in size of plastoquinone (PQ) pool
(Fv /Fo ) (Fig. 2C) and (ii)
hindrance in the movement of electron from PSII to the PQ pool thereby
resulting into accumulation of a strong fluorescence quencher:
P680+ (Govindjee, 1995), as revealed
by decline in the Area (area over the fluorescence curve), which
ultimately reduced maximum quantum yield for primary photochemistry
(ФPo ) (Fig. 2C). Therefore, reducedФEo or Phi_Eo (yield of
electron transport) and Ψo orPsi_o (efficiency to move the electrons of
trapped exciton into ETC beyond QAˉ) were obtained, that
leads to decreased QAˉ pool size on PSII acceptor side,
thereby signifying the obstruction in electron flow from PSII to PSI and
limitation in QAˉ reoxidation
(QAˉ‒QA) rate, which is confirmed by the
increased Fo/Fm values in the
present study that actually reveals that QAˉ reoxidation
(QAˉ‒QA) rate was much lower than its
reduction rate by QB. Ni2+-stressedAnabaena cells, showed little changes in the values ofΨo ; however, yield of primary electron transport
(ФPo ) and its product yield of electron transport
(designated by ФEo ) were sharply decreased in
comparison to control thereby indicating that hindrance in electron
transport was higher due to light dependent reaction (designated byФPo ) than dark reaction beyond
QAˉ (Ψo ), which collectively
suggests that Ni2+ might have hampered the primary
charge separation by the replacement of Mg2+ in the
antennas’ chlorophyll and PSII RCs as discussed by Kupper et al. (2003).
Furthermore, the drop in above parameters was extreme when Ni+SA treated
cyanobacterial cells were supplemented either with c-PTIO
(Ni+SA+Ca+c-PTIO), EGTA (Ni+SA+NO+EGTA) or both (Ni+SA+c-PTIO+EGTA),
suggesting that SA in absence of Ca2+ or/ and NO was
unable to counteract Ni2+-toxicity.
Ni2+-induced reduction in fluorescence yields ratio
for open and closed states
(Fm/Fo ) signifies the destructive
effect of the Ni on the structural integrity of PSII RCs. Further, the
significant increase in Sm values upon treatments
of Ca2+ and NO scavengers denotes that PQ
heterogeneity fasten the electron transfer capacity as well as
QA reduction on PSII acceptor side, thereby explaining
that these treatments hampered the total electron accepting capacity
(Ghassemi-Golezani and Lotfi, 2015).
Further, upon c-PTIO or/ and EGTA treatment to
Ni2+-stressed Anabaena cells, the balance
between active/ inactive RCs was disturbed, and the greater load might
have been experienced by active RCs
(Fv/Fo ); therefore, increased
values for energy flux parameters i.e. ABS/RC,
TRo/RC, ETo/RC andDIo/RC were noticed in the present study (Fig.
2C). These active RCs, absorbed the available light but were not able to
collect the huge excitation energy and convert into ‘energy sink’
thereby dissipating most of the energy in the form of fluorescence/ heat
as advocated by ФPo(Fv/Fm ) values; therefore, under
similar conditions, the ABS/RC values were increased as limited
number of active RCs increased the RC turnover rate to meet the complete
reduction of PQ pool (N ). The increment inTRo/RC (QA‒QAˉ)
values upon c-PTIO or/ and EGTA treatment to
Ni2+-stressed test organism, might be due to
inhibition in QAˉ reoxidation
(QAˉ‒QA), which hampered the electron
transfer efficiency to QB. The increasing values for
dissipated energy flux (DIo/RC ) and the quantum
yield of energy dissipation (ФDo ) upon
Ca2+ or NO scavenger supplementation to
Ni2+-stressed cells, justified that in order to
maintain the equilibrium between absorption and consumption of energy,
excess excitation energy was transformed into thermal dissipation as
argued earlier by Wang et al. (2012); which drastically decreased the
overall performance of PSII (PIABS ) (Fig. 2C).
On the other hand, SA, CaCl2 and SNP supplementation to
Ni2+-stressed culture medium repaired the structural
integrity of PSII by decliningFo/Fv and improvingFv/Fo ,Fm/Fo andФPo values. The Fo values
got lowered due to which increment in Fv was
observed which suggested towards betterment of PSII acceptor side
(Ghassemi-Golezani and Lotfi, 2015). Upon SA, CaCl2 and
SNP application, the recovery in Fm values
elaborated that either they might have bring the conformational changes
in D1 protein of PSII by modifying the properties of electron acceptors
(Andréasson et al., 1995) or might improve the Mn2+and OEC extrinsic proteins, which impeded electron donation from
H2O to PSII (Najafpour et al., 2012; Kalaji et al.,
2016) and ultimately the PSII activity. While working on heat-stressed
tall fescue plants, Chen et al. (2013) have suggested that NO either
might have improved the gene expression of PSII complex likepsbA , psbB and psbC or speeded electron
transportation from OEC to D1 protein. The possibility behind the
Ca2+-induced recovery in
Ni2+-stressed Anabaena cells could be: (i)
improvement in the pigment contents, which actively participates in the
assembly of PSII to support the proper functioning (Zakar et al., 2016),
and (ii) improvement in intracellular
Ca2+-accumulation, which is essential cofactor for
water oxidation, the Ca binding sites in PSII, and the transition of all
S-state, as was assumed by Najafpour et al. (2012). In addition, a steep
decline in ABS/RC , TRo/RC ,ETo/RC and DIo/RC values
under similar conditions, justified that PSII complex was enough capable
to handle the energy equilibrium between absorption and utilization of
light energy by active PSII RCs thereby minimizing the potential for
photo-oxidative damage; which finally improved the overall performance
of PSII as higher PIABS values were observed in
the present study (Fig. 2C).
The significant increment in respiratory rate upon c-PTIO or/ and EGTA
application to Ni2+-stressed Anabaena cells
(Fig. 2B) was probably to meet out the basic demand of ATP to carry out
basic metabolism of cell (Tiwari et al., 2019a), as photosynthetic
electron transport activities were hampered by reducing ATP supply under
similar conditions (Fig. 2A,C).