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).