Salicylic Acid Induced Physiological Responses of Microchaete Sp. NCCU-342 Under PQ Stress

Weeds are the worst category of agricultural pests as they compete with crops for water, nutrients, and light, grow faster than crops and eventually reduces the crop productivity thereby increasing the production cost. Herbicide has been used as a management tool to control weeds, by modifying their physiological activities like reducing photosynthesis, increasing protease activity and free radicals etc. The application of non-selected herbicides kills not only the weeds but also non-target organisms including the main crop. Herbicides like paraquat have been used in rice elds but its toxic effects on other organisms is at par. Salicylic acid, a plant growth regulator, is known to regulate oxidative stress in plants subjected to unfavourable environmental conditions. Given this, the present study was designed to study the effect of salicylic acid in Microchaete sp. NCCU-342 exposed to paraquat. The results obtained demonstrated that the paraquat toxicity elevated MDA and H 2 O 2 levels, indicating free radical formation. However, supplementation of salicylic acid in the culture medium reduced the paraquat toxicity. The content of MDA and H 2 O 2 in presence of salicylic acid was similar to the control. It enhances phenolic compound (29.07%) and phytohormones content (IAA-2.96%, SA-96.59%) and regulate the redox state of the cells. A signicant change (recorded in percentage) was observed in the antioxidant enzyme activities including SOD (121.62 %), CAT (30.38 %), APX (74.38 %), GPX (127 %) and GR (141%) in presence of paraquat and after supplementation of salicylic acid. Antioxidant assay conrmed the change in antioxidants in form of phenolic compounds. Salicylic acid supplementation also enhanced the antibacterial activity of cyanobacteria, highlighting that it induces resistance against biotic stress as well as the abiotic stress. This work provides evidence for the ability of salicylic acid to alleviate the paraquat-induced toxicity, maintain redox state and resist the cell against both biotic and abiotic stress.


Introduction
Herbicide through increase crop yield but their residual toxic impact on both selective and non-selective target, in long run disrupts the ecological balance between soil, crop and micro ora 1 . Paraquat (PQ) is a very well-known non-selective herbicide that has been widely used in agriculture elds such as Wheat, Rice, Maize, Cotton, Potato and has shown high toxicity against plants, animals and humans 2 . PQ acts by reducing the production of NADPH in a photosynthetic organism. It is an oxidant that interferes with the electron transfer reaction by accepting the electron from photosystem I and transferring it to molecular oxygen and producing reactive oxygen species (ROS) and reducing the formation of NADPH 3 .
Accumulation of ROS leads to lipid peroxidation and induces membrane damage, breakdown of photosynthetic apparatus, ion leakage, eventually leading to cell death and overall reduction in the crop Cyanobacteria are the pioneer photosynthetic bacteria that are known to play an important role in sustainable agriculture in rice ecosystem. Nostoc muscorum Anabaena sp., Calothrix, Microchaete sp. are classi ed as biofertilizers as they x nitrogen, release plant growth promoter substances such as phenols, phytohormones, and increase the water holding capacity of soil 6 . Application of PQ in rice eld is known to effect cyanobacteria that is used as biofertilizer 7 .
Salicylic acid (SA) is an important signalling molecule released by plants under stressful conditions 8 . It is an endogenous plant hormone that modulates metabolic and physiologic response under oxidative stress 9 as induced by salinity 10 , heavy metal 11 , drought 12 , heat 13 , cold 14 and herbicide 3 . It has been reported that exogenous application of salicylic acid also reduces the toxicity of herbicide by modifying the antioxidant enzyme activity 3 . SA is known to control the pesticide effect such as H.A.M-45, Termite kill, Anchor in Green gram 15 , Napropamide in rapeseed and mustard plants 16 and Clethodium in maize 17 . In barley plant under paraquat toxicity, the treatment of salicylic acid-induced the expression of SOD, CAT, APX, and POD 3 . Exogenous application of SA is known to reduce the quinclorac (herbicide) toxicity by modulating the antioxidant defense enzymes such as SOD, CAT, APX, GPX and GR 18 . SA also elevates the content of non-enzymatic antioxidants such as ascorbate and glutathione which are directly involved in the detoxi cation of ROS and also play an active role in ascorbate-glutathione cycle to maintain the redox state 19 . Glutathione performed the redox couple reaction with nicotinamide adenine dinucleotide phosphate and provides stress resistance to the plants 20 . Supplementation of SA in wheat seedlings showed the increased content of ascorbate, glutathione and other related enzymes 21 .
To validate our hypothesis that SA helps in maintaining the cellular redox state of cyanobacteria under PQ stress. "Cyanobacteria culture treated with PQ was grown in presence and absence of SA for 96 hours. The endogenous phytohormones (IAA and SA), phenolic content, redox biomarker (GSH and GSSH and its ratio), antioxidant enzymes, DPPH (for free radical scavenging potential) and anti-bacterial activity (for biotic potential) were evaluated as these parameters re ect the tolerant potential of the organism.

Materials And Methods
Culture maintenance. Microchaete sp. NCCU-342 were procured from IARI, New Delhi and grown in BG-11 medium without sodium nitrate illuminated with the uorescent light with 2000 ± 200 lux intensity for 12:12 light/dark cycle under the aseptic culture room 54 .
Growth of Microchaete sp. NCCU-342 in presence of PQ and SA. Microchaete sp. NCCU-342 was grown in presence of PQ (0, 0.1, 0.2, 0.4, 0.6, 0.8, 1.0, 1.2 µM) to determine its tolerance limit. After 96 hours, biomass was washed twice with distilled water, harvested by centrifugation (5000 rpm for 10 minutes) and dried at 60 ℃. Growth of the organism was check by the chlorophyll content. Total chlorophyll was analyzed according to the modi ed protocol of Mackinney, (1942) 55 . Dried biomass was dissolved in 95 % methanol (10 ml) and incubated at 65 ℃ for 30 minutes in a water bath, centrifuged at 5000 rpm for as blank. Total chlorophyll was calculated by using the following formula.
Tolerance limit of PQ was determined through linear regression 56 . Further, at 50 % inhibitory concentration, 0.3 mM of SA was supplemented to check the physiological parameters i.e., endogenous phytohormones (IAA and SA), phenolic content, redox biomarkers (GSH and GSSH and its ratio), antioxidant enzymes, DPPH (for free radical scavenging potential) and anti-bacterial activity (for biotic potential).
Determination of Malondialdehyde and hydrogen peroxide (supplementary data). Malondialdehyde (MDA) and hydrogen peroxide was estimated as done in our previous study 57 .
Determination of total Phenol content. The total phenolic contents were estimated by using protocol of Singleton and Rossi (1965) 58 . Dried biomass (50 mg) was homogenized in 80% methanol (10 ml). Distilled water (5 ml) and Folin Ciocalteu's reagent (0.5 ml) were added to the methanolic extract (40 µL) and kept for 5 minutes. Then, 20% sodium carbonate (1.2 ml) was added and the volume of the test sample was adjusted to 10 ml with distilled water and allowed to stand in dark at room temperature for 60 minutes. Absorbance was recorded at 750 nm after the development of intense blue colour. This equation was used to calculate the amount of standard gallic acid with the regression coe cient (r 2 ) being 0.9666.
Determination of endogenous indole acetic acid (IAA) and salicylic acid (SA) content. The protocol of Ehmann (1977) 59 was used to determine the IAA content produced. A colorimetric technique was adopted by using Salkowski reagent (one ml of 50 mM ferric chloride dissolved in 35 % perchloric acid). Biomass (50 mg) was centrifuged at 10,000 rpm for 15 minutes at 4°C. The supernatant was collected and ltered by Millipore lter, 0.45 µm and acidi ed with 1.0 M hydrochloric acid. Extraction was done by partitioning with ethyl acetate (1:3, v/v; thirce) and evaporated using Rotary evaporator (Buchi type). Dried extract was dissolved in 95% methanol (1 ml). Salkowaski reagent (1 ml) was added to this extracted methanolic solution (0.5 ml) and kept in dark. Development of the red-pink colour indicates the presence of IAA and the absorabnce was taken at 530 nm, against Salkowski reagent as blank. Standard IAA (1 mg/ ml) was prepared as stock solution. It was further diluted with distilled water to obtain different working concentrations (0.5 -5 µg/ml). The calibration curve was described by the equation given below with the regression coe cient (r 2 ) being 0.9779. This equation was used to calculate the amount of standard IAA.
Endogenous salicylic acid production was also checked. Salicylic acid extraction was done by using the protocol of Raskin et al. (1989) 60 and quanti cation was done by following the protocol of Warrier et al. 61 . For extraction of endogenous salicylic acid, 50 mg biomass was homogenized with 90% methanol (10 ml) and centrifuged at 5000 rpm. Pellet was dissolved in absolute methanol (10 ml) and centrifuged at 8000 rpm for 15 minutes. The supernatants were evaporated at 60°C and the residue was re-suspended in 5 % trichloroacetic acid (5ml) and centrifuged at 5000 rpm. The supernatant was partitioned twice with double the volume of ethyl acetate 60 . The production of salicylic acid was con rmed by simple colorimetric test. To the ethyl extract (1 ml), alcoholic ferric chloride (1 ml) was added, formation of purple-violet colour indicated the presence of salicylic acid that was measured at 540 nm. Salicylic acid stock solution (1mg/ml) was further diluted with distilled water to obtain different working concentrations (0.01-0.5 µg/ml). Absorbance was taken at 540 nm. The calibration curve was described by the equation given below with regression co-e cient (r 2 ) being 0.9582. The following equation was used to calculate the amount of standard salicylic acid.
Where Y is absorbance at 540 nm and X is concentration of salicylic acid in µg/ml.
Determination of reduced and oxidized glutathione (GSH) and redox state. Glutathione was determined by the method of Anderson, (1985) 62 . Fresh biomass (50 mg) was homogenized in 5 % sulfosalicylic acid and centrifuged at 10,000 rpm for 10 minutes under cold condition. After centrifugation, in 500 µl supernatant was added to 600 µl of phosphate buffer (100 mM, pH 7.0) and DTNB (40 µl) and incubate for 2 minutes at room temperature. After incubation absorbance was read at 412 nm. For oxidized glutathione, the GSH was masked by derivatization with 2-vinylpyridine. Reduced glutathione was calculated by subtracting the value of oxidized glutathione from the total glutathione. The redox state was calculated as the ratio of GSH to oxidized glutathione (GSSG).
SOD, CAT and APX activity was estimated by the method as followed in our previous study 57 Determination of Glutathione Reductase (GR) activity. GR activity was estimated by the method of Asgher

Concluding remarks
Paraquat shows toxic effect to the non-target cyanobacterium, Microchaete sp. NCCU-342 by changing in their normal biochemical and metabolic activity. PQ increases oxidative damage through free radical formation. Supplementation of salicylic acid improves the antioxidant machinery, increases phenolic compound and phytohormones that ultimetly reduces the free radical level. Salicylic acid application also enhanced the antimicrobial activity of the organism. Supplementation of salicylic acid not only counteracts the toxic effect of herbicide through managing the antioxidant potential but also make it tolerant against biotic stress. Therefore, for the sustainable agriculture, the formulation of salicylic acid with cyanobacterial biofertilizer would be a better option.

Results And Discussion
During last few decades, treatment of herbicide has been adopted for better crop production. The nonselective herbicides like paraquat, glufosinate, basagran, and glyphosate not only affect the weed but also change the physiology of non-target organism like main crop and biofertilizer i.e., cyanobacteria 22 .
PQ (methyl viologen) is a cationic herbicide that disturbs electron transport by accepting the electron from photosystem I. Cyanobacterial rice bio fertilizers like Microchaete sp. is also highly sensitive to PQ due to presence of photosystem I 23  For comparative growth analysis, Microchaete sp. were grown for 96 hours in presence of only PQ (0.6 µM), and SA supplemented (0.3 mM) PQ exposed cultures. After 96 hours, the growth was found maximum in control. However, growth declined in PQ treated culture (50%) whereas supplementation of SA in the PQ treated culture showed higher growth (35%) (signi cant at P ≤ 0.05). Supplementation of exogenous SA in Microchaete sp. culture showed good growth than the PQ exposed cultures (Fig. 1b). The growth enhancement by the application of salicylic acid has been also reported in Chlorella vulgaris 29 .
Chlorophylls represent the fundamental part of the whole metabolism of the photosynthetic organisms. Increase in chlorophyll content re ect growth, whereas decrease in chlorophyll re ect the unfavourable condition for growth. Under confocal microscopy, chlorophyll was used as a natural marker. Microchaete sp. NCCU-342 grown for 96 hours with 0.6 µM PQ exhibited degradation of chlorophyll. Normal healthy cells of control sample were more uorescent that con rmed growth. PQ reduced the chlorophyll content and resulted in poor growth that decreased the uorescent whereas supplementation of salicylic acid improved chlorophyll and reduced the membrane damage and lysis, and gave higher uorescent (Fig. 1cf). Phormidium corallyticum under black band disease 30 and in Synechococcus elongatus PCC 7942 also showed reduced uorescence under hydrogen peroxide stress. 31 Effect of PQ and SA + PQ on oxidative stress under PQ stress. PQ induced the production of free radicals MDA and hydrogen peroxide (Fig. 2a-b). MDA level increased by 8.16 % in PQ exposed culture as compared to control and reduced by 15.63 % in culture having both SA and PQ as compared to PQ treated culture (signi cant at P ≤ 0.05). As compared to control, the level of hydrogen peroxide in PQ treated set increased by 4.11 % and reduction in hydrogen peroxide level was found as 2.455 % in culture with both SA and PQ (signi cant at P ≤ 0.05). PQ (di-cation) disrupts the electron transport system and form the mono-radical cation. This mono-radical cation interacts with molecular oxygen to produce other toxic radical such as O2 •− and hydrogen peroxide 32  Effect of PQ and SA + PQ on antioxidant enzymes under PQ stress. The toxicity of PQ causes the generation of free radicals. In order to counteract these free radical's antioxidant enzymes like SOD acts as the rst-line defense but produces hydrogen peroxide as its end product that gets eliminated by CAT, APX and GPX 32,38 . As compared to control, a 78.37 % increase in SOD activity was recorded in PQ treated culture set. Supplementation of salicylic acid in PQ-treated culture was found to increase the activity by 121.62 % as compared to control (Fig. 3a). Similarly, CAT (46.38 %), APX (16.73 %), GPX (240 %) and GR (15%) activity also increased in PQ treated culture as compared to control, but their induction was enhanced up to 30.38 % in CAT, 74.38 % in APX, 127 % in GPX and 141% in GR after the supplementation of salicylic acid in PQ culture set as shown in Fig. 3b-e (signi cant at P ≤ 0.05).
PQ stimulate the enzyme activity of SOD, CAT, APX GPX and GR in Azolla-Anabaena symbiotic system 33 and in Chlorella vulgaris 2 . Accumulation of hydrogen peroxide plays an important role as signalling molecule and activates other set of enzymes i.e., CAT GPX and APX. CAT and GPX that catalyse the dismutation of hydrogen peroxide into water and oxygen. APX uses ascorbate as reducing power and potentially catalyze the removal of superoxide and hydrogen peroxide and GR accumulates GSH from GSSH and ascorbate for ascorbate peroxidase in ascorbate-glutathione cycle 5 .
Salicylic acid enhanced the activity of SOD, CAT, APX GPX and GR in the present study. Salicylic acid regulates the antioxidant enzymes under stressful condition and maintain the redox state of cell Glutathione is a tri-peptide non-enzymatic antioxidant compound that operates in cellular detoxi cation and maintain the redox homeostasis of the cell. It occurs in two form reduced glutathione (GSH) and oxidized glutathione (GSSH) 20 . GSSH further gets reduced by glutathione reductase. This cycle regulates the redox homeostasis of the cell 40 . The reduced glutathione (GSH) was decreased in PQ treated culture by 25.29 % as compared to control. The culture supplemented with both SA and PQ showed 67.42 % increased of GSH as compared to control. The GSSH increased by 56.79 % in PQ treated culture as compared to control, whereas 80.69 % reduction was recorded as compared to PQ treated culture. The ratio of reduced glutathione to oxidized glutathione was lowered in PQ treated culture by 20.94 % as compared to control and increased in SA + PQ culture by 28.02 % as compared to PQ treated culture (Fig. 4a-c). The activity of glutathione reductase depends on NADPH, and PQ blocks the regeneration of NADPH from NADP + which may be the reason of reduced GR activity which in turn reduced its activity and destabilizes the redox state. PQ has been also known to reduce the glutathione in cucumber 41   The antibacterial activity was evaluated by using aqueous extracts of each culture against Pseudomomas aeruginosa. The diameters of the zone of inhibition was recorded in millimetres (Fig. 5b). Control sample showed no zone of inhibition but PQ treated samples (60 and 80 µg/ml) exhibited zone of inhibition (9.6 mm and 11 mm diameter). SA supplementation in PQ exposed culture showed zone of inhibition in all tested concentration of extract (20,40,60, 80 µg/ml) that increased in a dose dependent manner (10 mm, 10.4 mm, 16.5 mm and 20 mm). Zone of inhibition by streptomycin was recorded as 10.5 mm, 10.8 mm, 20 mm and 20.2 mm from lower to higher concentration respectively. Thus, the best anti-bacterial activity was found in presence of SA + PQ. Treatment of salicylic acid in rosemary also showed antibacterial activity under salinity. The enhanced phenolic and endogenous IAA and SA content and antioxidant compound by salicylic acid supplementation may be the reason for the enhancement of antioxidant and antibacterial activity. In this study, exogenous application of salicylic acid to PQ exposed cultures alters the antioxidant activities. The antioxidant and antibacterial activity results showed that SA supplementation not only resist abiotic stress but also provide resilience against biotic stress. These scavenging activities showed that SA modulates antioxidant activity. The antioxidant and antibacterial activity showed the tolerance ability of Microchaete against PQ after SA supplementation.
Effect of PQ and SA + PQ on phenolic compound and phytohormone of Microchaete sp. under PQ stress.
Accumulation of phenolic compounds was found in all cultures. As compared to control, PQ-treated culture showed an 8.85 % increase in total phenolic content (signi cant at P ≤ 0.05). Supplementation of salicylic acid showed a 29.07 % increase in total phenolic content as compared to PQ exposed culture (signi cant at P ≤ 0.05). SA induced the production of phenolic compound in culture with both salicylic acid and PQ as observed in our study (Fig. 6a). Due to different environmental stress, plant produces secondary metabolites such as phenolic compounds, terpenoids, avonoids, etc. for protection. These metabolites have high antioxidant potential and play a major role in defense against stress. Radwan.
(2012) 17 , reported that salicylic acid induced the phenolic compound in clethodium herbicide stressed maize to enable it to survive.
The endogenous phytohormones (IAA & SA) were also induced in all sets (Fig. 6b-c). The endogenous level of IAA increased by 13.43 % in PQ as compared to control, whereas the supplementation of SA in PQ exposed culture resulted in further increase by 2.96 % only as compared to PQ treated cultures. The amount of endogenous salicylic acid also increased in PQ treated culture by 96.59 % as compared to control. The supplementation of salicylic acid with PQ resulted in higher SA accumulation by 4.72 % as compared to PQ treated culture (Fig. 6c). Exogenous supplementation of SA in higher plants induced endogenous SA content e.g. in cucumber under salt stress 31