Exogenous Phytohormones Modulates Cypermethrin Stress in Anabaena sp. and Nostoc Muscorum: Toxicity Alleviation by Up-regulation of Ascorbate-glutathione Cycle


 Present study demonstrated the effect of phytohormones KN and IAA under cypermethrin (Cyp1; 2 µg ml− 1 and Cyp2; 4 µg ml− 1) toxicity in two nitrogen fixing cyanobacteria Nostoc muscorum ATCC 27893 and Anabaena sp. PCC 7120 by investigating growth, exopolysaccharides (EPS) contents, protein content, oxidative stress visualization inside the cell and ascorbate- glutathione cycle. Decline in growth were noticed under both the doses of cypermethrin but the decline was more (30%) at higher dose in Anabaena sp. PCC 7120 as compare to N. muscorum. This decrease was due to increased production of oxidative biomarkers (i.e. SOR and H2O2) subsequently membrane got damaged which was noticed by measuring MDA equivalents content (in vivo visualization). Kinetin and IAA alleviated the SOR and H2O2 content resulting in recovery of cellular membrane and the growth was optimized up to control level. Detoxification of H2O2 is guided by enzymes/metabolites of AsA-GSH cycle like ascorbate peroxidase (APX), glutathione reductase (GR), monodehydroascorbate reductase (MDHAR) and dehydroascorabte reductase (DHAR) activity were found to be stimulated at lower dose of cypermethrin as compare to higher dose. while the amount of metabolites: total ascorbate (AsA), total glutathione (GSH) and ratios of reduced/oxidized AsA (AsA/DHA) and GSH (GSH/GSSG) showed significant reduction at both the doses but the reduction was more at higher dose of cypermethrin. Kinetin and IAA positively regulate the AsA-GSH cycle by enhancing the activity of APX, GR, MDHAR and DHAR activity and raising the metabolites content and their reduced/oxidized ratio. This study suggests the increased enzymatic activity and AsA/DHA ratio leads to reduced production of H2O2 in the presence of both the phytohormones which further leads to enhanced growth in both the organism but the effect of KN and IAA was more pronounced in N. muscorum suggesting its resistivity against stress.


Introduction
Indiscriminate application of pesticide in agriculture badly affects the growth of plants and cyanobacteria by generating reactive oxygen species (ROS) [1][2][3] . Excessive production of ROS, such as the superoxide radical (O 2 •− ) and H 2 O 2 results a toxic state called oxidative stress 4 .Their generation are known by various electron transport systems and thus increased the chance of damage of large biomolecules like lipids, proteins and DNA 3,5,6 . On the other hand, ROS in cell is now being con rmed as secondary messenger and guide several physiological processes via redox signalling and regulate the protein and gene expression 7,8 . Defence machinery having numerous antioxidant systems evolved in cells to control the amount of ROS and minimizes the damage caused to macromolecules and involve them in signalling pathway. Defence system comprises both enzymatic and non-enzymatic antioxidants and among antioxidants, ascorbate-glutathione (AsA-GSH) cycle play a prominent role to control/detoxify the ROS [9][10][11] . It enclosed three independent redox couples: reduced and oxidized ascorbate ratio (AsA/DHA), reduced and oxidized glutathione ratio (GSH/GSSG), and NADPH/NADP + 6,12-14 and enzymes i.e., ascorbate peroxidase (APX), glutathione reductase (GR), monodehydroascorbate reductase (MDHAR) and dehydroascorbate reductase (DHAR) 12,15-17 . These enzymes are responsible for redox cycling of AsA and GSH. By the involvement of these enzymes, the reduced and active forms of ascorbate and glutathione are maintained at optimal level and consequence of this AsA-GSH cycle operated well inside the cell to minimize the toxicity caused by excess ROS 3,17,18 .
Cyanobacteria are essential constituents of the fresh water ecosystem and paddy eld soil where moist and nutrient rich environment regulate their growth, physiology, abundance and habitat selection 19,20 . The indirect effect of pesticide also recorded on cyanobacteria by notifying the oxidative stress caused by overproduction of free radical 17,21,22 . Apart from having several defensive processes, they are known to regulate cellular oxidative stress by operating AsA-GSH cycle including few enzymes under pesticide as well as metal stress condition 23,24 .
Phytohormones are known to regulate the growth of plants as well as cyanobacteria under normal as well as stress condition 25,26 . They provide signals against several stress conditions and allow plants to survive by adopting crucial strategies to counteract the adverse effects of environmental stresses [27][28][29][30] .
Cyanobacteria were also reported to produce several phytohormones [31][32][33][34][35] . In plants, effect of phytohormones on regulating the oxidative stress were highly discussed by AsA-GSH cycle 36-39 but in cyanobacteria, it is still a matter to unrevealing the mechanism. Therefore, present study focused on the potential role of KN on regulating the oxidative stress generated by pesticide via AsA-GSH cycle in Anabaena PCC 7120 and Nostoc muscorum.

Result
Phytohormones regulated growth of cyanobacteria content but it was still less than that of untreated control. Under similar condition the level of protein content in N. muscorum was almost parallel to the control sample ( Fig. 1).

Phytohormones regulated EPS content in cyanobacteria
Results pertaining to exopolysaccharides (EPS) content are represented in gure 2. Upon cypermethrin treatment (Cyp 1 and Cyp 2 ) the content of exopolysaccharides was found to decline by 14% and 33% in Anabaena sp., and by 10% and 28% in N. muscorum, respectively over the value of controls.
Exogenous phytohormones KN/IAA enhanced the content of EPS appreciably in both the cyanobacteria exposed to Cyp 1 stress over the values recorded in controls. Both the phytohormones in Cyp 2 stressed tested cyanobacteria, though showed increasing trend in EPS content but it remained less than that obtained in untreated controls. Phytohormones modulated enzymatic activities of AsA-GSH cycle: APX, GR, DHAR and MDHAR Results pertaining to the enzymes of ascorbate-glutathione cycle are represented in gures 4 and 5. In cultures of Anabaena sp. and N. muscorum exposed to Cyp 1 stress, the activity of APX was found to accelerate by 5% and 11% while after Cyp 2 treatment the activity of this enzyme suppressed by 8% and 5%, respectively as compare to respective controls. Addition of KN to cypermethrin stressed cultures exposed to Cyp 1 dose caused further enhancement (16% and 25%) in the activity of APX in Anabaena sp. and N. muscorum, respectively. Kinetin supplementation to Cyp 2 stressed cultures improved the activity of APX which was parallel to the activity of control in both the cyanobacteria (Fig. 4). Almost similar pattern in the activity of APX enzyme in both the cyanobacteria exposed to cypermethrin (Cyp 1 and Cyp 2 )

Phytohormones regulated ROS content in cyanobacteria
was recorded with the addition of phytohormone IAA (Fig. 5).
Activity of GR, DHAR and MDHAR (Figs 4 and 5) showed similar trend in both the treated cyanobacteria but at Cyp 2 dose their activity increased as compare to control. Above result mentioned that both the hormone e ciently expressed its role in Nostoc muscorum.
Phytohormones optimized ascorbate (AsA) and glutathione (GSH) level Total ascorbate (AsA+DHA) and AsA/DHA ratio were also quanti ed under Cyp and KN and IAA treatment (Table 1 and 2). There was a decrease in total ascorbate (T-ASA) content both the doses of cypermethrin in both the tested organisms. Under similar condition, reduction was more pronounced in AsA, so that AsA/DHA ratio also decreased. However, under the treatment of KN and IAA, AsA-GSH pool restored as compared to the samples treated with cypermethrin.
Likewise ascorbate, total glutathione (T-GSH) and GSH/GSSG ratio showed similar trend in the presence of KN and IAA (Table 3 and 4). However, GSH content declined more as compared to AsA content at both the doses in both the cyanobacteria. In the above mentioned result, N. muscorum is more expressive for KN and IAA.

Discussion
In current study decline in growth at both the doses of cypermethrin (Cyp 1 : 2 µg ml -1 and Cyp 2 : 4 µg ml -1 ) (Figs. 1 and 2) was noticed in both the cyanobacteria which might be due to enhanced production of ROS causing membrane damage (Fig. 3). Tiwari and Prasad 40 noticed the decline in growth due to decline in physiological activity and defense mechanism in cyanobacteria (Anabaena PCC 7120 and N. muscorum). This decline was mitigated by the phytohormones (KN and IAA). Similar results were noticed by Tiwari et al. 41 in the organism N. muscorum in the presence of KN. Protein is also one of the important indicators to assess the growth of organism under stress conditions. In this study protein content increased at lower dose of cypermethrin ( Fig. 1) representing its defensive strategy which is concurred with increased activity of enzymatic antioxidants (Tables 1 and 2 Effect of oxidative stress was also correlated with the status of metabolites: ascorbate (AsA and DHA, AsA/DHA) and glutathione (GSH and GSSG, GSH/GSSG) involved in AsA-GSH cycle. These metabolites play integrative role in scavenging the ROS and prevent the cell from oxidative stress. Ascorbate (AsA) is known to quench ROS either by direct or indirect way via generating other non-enzymatic antioxidants like alpha-tocopherol 12 . Dolatabadian and Jouneghani, 48 showed the effective role of exogenously supplied ascorbate on activity of enzymatic antioxidants of NaCl-treated common beans. In this study, total ascorbate (AsA+DHA) and total glutathione (GSH+GSSG) contents decreased under cypermethrin (Cyp 1 and Cyp 2 ) treatment (Tables 1-4) which might have took place due to its inhibitory effect on their regeneration and/or excessive utilization of these compounds during the process of pesticide detoxi cation. Similarly, AsA/DHA and GSH/GSSG ratio decreased at both the doses of cypermethrin justifying the decreased AsA-GSH pool leading to oxidized environment inside the cyanobacterial cells. Decline in AsA and GSH content might de due to pesticide mediated hindrances in their biosynthesis.
After KN and IAA treatment to the tested organisms, considerable enhancement in AsA and GSH pool supported the cells in combating the cypermethrin induced toxicity which further tends to reduction in H 2 O 2 content (Fig. 3). In this study, we found that KN and IAA controlled the ascorbate-glutathione

Measurement of growth
The cell culture (100 ml) was harvested, centrifuged at 4000g for 10 min, then washed with distilled water thrice and oven dried at 80˚ C for 48 h and weighed using digital weighing balance (Contech-CA 223, India).

Measurement of EPS
Extraction and estimation of EPS content were done by the method of Sharma et al. 42 . For extraction of the cyanobacterial EPS, 100 ml of treated and untreated cyanobacterial cells was centrifuged at 3000 g and after separating the settled biomass, cell-free culture containing the EPS was taken. It was then concentrated tenfold by evaporation at 40 ˚C, washed with isopropanol two-three times to remove any contaminants, and then nally dried at 37 ˚C and then analyzed for glucose.

Measurement of protein
Protein content was estimated by the method of Lowry et al. 49 modi ed by Herbert et al. 50 . The amount of protein was expressed as Protein (μg mL -1 culture). The amount of protein was assessed by using standard curve set with bovine serum albumin (BSA).

In vivo assessment of ROS production and lipid peroxidation
The histochemical visualization of O 2 •− , H 2 O 2 and MDA equivalents content was performed by in vivo staining of treated and untreated cyanobacterial cells with nitrobluetetrazolium (NBT; Sigma) and 3, 3′ diaminobenzidine (DAB) (Sigma) 51 and Schiff's reagent (Sigma) 52 , respectively. NBT stained deep blue formazan precipitate, DAB forming a reddish-brown polymerization product and MDA equivalents reaction product stained by Schiff's reagent. Thereafter, cells were imaged with Leica (Model: DM 2500).

Measurement of activities of APX, GR, MDHAR and DHAR
Ascorbate peroxidase (APX; EC 1.11.1.11) activity was determined by following the method of Nakano and Asada 53 . Decrease in the absorbance was determined spectrophotometerically at 290 nm and enzyme activity was calculated by using an extinction coe cient of 2.8 mM −1 cm −1 . One unit (U) of enzyme activity is de ned as 1 nmol ascorbate oxidized per minute.
Glutathione reductase (GR; EC 1.6.4.2) activity was assayed according to the method of Schaedle and Bassham 54 . Decrease in the absorbance was measured spectrophotometerically at 340 nm, and GR activity was quanti ed by using an extinction coe cient of 6.2 mM −1 cm −1 . One unit (U) of enzyme activity is de ned as 1 nmol NADPH oxidized per minute.
Monodehydroascorbate reductase (MDHAR; EC 1.6.5.4) activity in tested cyanobacterial cells was estimated by following the method of Hossain et al. 55 . Decrease in the absorbance due to oxidation of NADH was measured spectrophotometerically at 340 nm, and MDHAR activity was quanti ed by using an extinction coe cient of 6.2 mM −1 cm −1 . One unit (U) of enzyme activity is de ned as 1 nmol NADH oxidized per minute.
Dehydroascorbate reductase (DHAR; EC 1.8.5.1) activity was determined by following the method of Nakano and Asada 56 . An increase in the absorbance was measured spectrophotometerically at 265 nm, and DHAR activity was calculated by using an extinction coe cient of 7.0 mM −1 cm −1 . One unit (U) of enzyme activity is de ned as 1 nmol DHA reduced per minute.

Measurement of ascorbate and glutathione
The measurement of total ascorbate (AsA + DHA), reduced ascorbate (AsA) and dehydroascorbate (DHA) were carried out by following the method of Gossett et al. 57 . In this assay, reduction of Fe 3+ into Fe 2+ with ascorbic acid in acidic medium took place and formation of red chelate between Fe 2+ and 2,2′-bipyridyl occurs. Dehydroascorbate was determined by subtracting AsA from AsA + DHA. Ascorbate content was calculated by using standard curve prepared with l-ascorbic acid. Total (GSH + GSSG), reduced (GSH) and oxidized glutathione (GSSG) were quanti ed by the enzyme-recycling method of Brehe and Burch 58 . This method followed the chronological oxidation of GSH by 5,5′-dithiobis-2-nitrobenzoic acid (DTNB) and reduction of GSSG in the presence of NADPH and glutathione reductase. Oxidized glutathione was quanti ed by pre-incubating 1 ml of 1:20 diluted extract with 40 ml of 2-vinylpyridine for 1 h at 25 ± 2 °C. The pre-incubated samples were used for determination of GSSG content. The GSH content was measured by subtracting GSSG from GSH + GSSG using as standard curve prepared with GSH.

Statistical analysis
The results presented are the means of three replicates (n = 3) to con rm the reproducibility of the data.
Since the results showed normal distribution, comparison between control and treatment's means was carried out by using one-way ANOVA to test signi cance level (Duncan's multiple range tests, DMRT) at P < 0.05.

Conclusions
The Tables Table 1 Impact of KN (20 nM) on total ascorbate (T-ASA) contents and reduced/oxidized (ASA/DHA) ratio of Anabaena PCC 7120 and Nostoc muscorum exposed to cypermethrin (Cyp) stress. Control: -Cyp, Cyp 1 : 2 µg ml -1 and Cyp 2 : 4 µg ml -1 . Data are means ± standard error of three replicates (n = 3). Values followed by different letters within same column show signi cant difference at P<0.05 signi cance level according to the Duncan's multiple range test.