An effective in-gel assay protocol for the assessment of acid phosphatase (ACPase) isoform expression in the fungus Serendipita indica

Abstract

The induction of acid phosphatase (ACPase) in the mycorrhizal fungi is an adaptive survival mechanism to cope in a low-phosphate environment. A mycorrhizal fungi Serendipita indica can induce the ACPase enzyme and enhance the phosphate (Pi) level to the host plant through Pi-solubilization mechanism, both intracellular and extracellular (media) levels. The spectrophotometer technique has been widely and commonly used to measure the ACPase enzyme activity in all microorganisms and plants using pNPP as a substrate. However, this technique cannot be useful when studying the involvement of ACPase isoforms in Pi-solubilization. In this article, we developed a single method to identify and express the ACPase isoforms of S. indica that contribute to the Pi-nutrition in the plant. This is native-PAGE electrophoresis with the in-gel assay and staining to detect the isoforms of the ACPase enzyme. The dark red-brown color developed after staining indicates the non-denatured (native) ACPase enzyme. This method utilized a modified minimal media for the de-repression of P-responsive genes such as ACPases with minimum salt contamination in the samples. This method will be helpful for the characterization of secretory and intracellular ACPases in fungi.

Introduction

Phosphorus (P) is the second most essential macronutrient required for plant growth and development. It is present abundantly in the soil in bound form. However, the availability of phosphate (Pi) or inorganic Pi to plant is limited, which often results in P-starvation and reduces plant growth and yield. Symbiotic association between the plant and arbuscular mycorrhiza (AM) or endophytic root colonizing fungi play a crucial role in increasing the Pi level in the rhizosphere. AMF and root colonizing endophytes were efficiently contributing towards Pi-solubilization under Pi-starvation conditions. In the Pi-deficit state, ACPase enzymes were expressed and secreted by the root colonized fungus to release orthophosphate (free Pi) from bound phosphate and improve the Pi availability in soil solution (Wang et al. 2017; Alvarez et al. 2012; Nguyen and Saito 2021). In in vitro conditions, endophytic fungi can recover Pi availability either via lowering the pH, secreting the organic acid, chelation of P with metal salts (Fe³⁺, Al³⁺, Ca²⁺) in the external environment, or via activating the synthesis and enhanced secretion of ACPase enzyme to release the Pi from bound P source (Etesami and Jeong 2021).

ACPase (EC number 3.1.3.2) is a well-known enzyme involved in the Pi-solubilization (Rejsek et al. 2012; Behera et al. 2017; Rombola et al. 2014; Mohd et al. 2019), which hydrolyses the phosphomonoester bond and release free Pi from bound P source. The role of ACPases has been studied in various mycorrhizal fungi (Alvarez et al. 2012; Nguyen and Saito 2021; Wang et al. 2017), including Glomus intraradices, G. claroideum (Joner and Johansen 2000), Rhizophagus clarus (Sato et al. 2015, 2019) Saccharomyces cerevisiae, Candida parapsilosis, C. albicans, and Aspergillus fumigates (Freitas-Mesquita and Meyer-Fernandes 2014) in P-deficient state. Intracellular and extracellular ACPase enzyme activity was studied earlier in various fungi because of its potential role in Pi-solubilization (Nahas 2015; Yadav and Tarafdar 2003; Della Mónica et al. 2018; Tarafdar et al. 2002; Gaind and Nain 2015). However, its isoforms distribution in the fungal cell is limited to a few fungi. S. cerevisiae, three isoforms of ACPase were identified (Krasnopevtseva et al. 1986). Similarly, ACPaseI and ACPaseII were identified, purified, and characterized in Trichoderma harzianum (Souza et al. 2016). Generally, secretory isoforms participate in extracellular Pi-solubilization, and intracellular ACPase participates in Pi- metabolism.

A root-endophytic fungi Serendipita indica (S. indica), is a growth promoter to their colonized host plant under extreme physical and physiological stresses. S. indica has the potential to colonize with a broad range of host plants (both dicot and monocot) by regulating the plant growth hormone. Additionally, it can also protect the host plants from biotic and abiotic stresses. Root colonization by S. indica during stress conditions was studied in many plants. The beneficial impact of S. indica colonization on to host was well documented. This list includes its role as a biofertilizer to promote nutrient mobilization, growth, yield, and bio-protector to overcome salt, acid, water tolerance, and heavy metal stress. S. indica also induces pathogen resistance (fungal and viral) in the host plant by activating the host antioxidative system (Gill et al. 2016; Anith et al. 2018; Mohd et al. 2017). Earlier studies have reported S. indica involvement in phosphate (Pi)-solubilization (Ngwene et al. 2016) and its transportation to their host.

In S. indica, ACPase enzyme activity can be measured by spectrophotometer, but native-acrylamide gel electrophoresis is required to identify the expression of ACPase isoforms. In this protocol, S. indica culture conditions, incubation period, and gel staining procedure have been modified and standardized to develop a method for identifying the ACPase isoforms and their expression from a single sample. Here, we provide a simple and efficient method that can also be used to analyze the expression of ACPase in axenic and monoxenic cultures.

Materials and methods

Fungal culture preparation

Serendipita indica culture was maintained on modified Hill–Kafer media (KF media) (Hill and Kafer 2001) with the following compounds (g/l); glucose (10), peptone (2), yeast extract (1), casamino acid (1), along with 50 ml/l of macroelements [KCl (10.4), NaNO₃ (120), MgSO₄⋅7H₂O (10.4), KH₂PO₄ (16.3) and K₂HPO₄ (20.9)] and 1 ml/l of microelement in [ZnSO₄⋅7H₂O (0.022), H₃BO₃ (0.011), MnCl₂⋅4H₂O (0.005), FeSO₄ (0.005), CoCl₂ (0.0016), CuSO₄⋅5 H₂O (0.0016), (NH₄)₆Mo₇O₂₄⋅4H₂O (0.0011) and Na₂EDTA (0.06)] from stock solution with 1 ml/l of vitamin stock solution, the media was sterilized at 121 °C for 40 min.

1 ml of 10⁶ spores of S. indica were inoculated in the 50 ml of KF media in 100 ml Erlenmeyer flasks and incubated at 32ºC for 72 h in a shaker (80–120 rpm).

Change of Media for ACPase induction and secretion

After the complete growth of S. indica in KF media, culture was washed with modified minimal media (MN media) 3–4 times (Table 1). After washing, the mycelia were transferred into MN media supplemented with 10 µM KH₂PO₄ (Pi), and the culture was incubated for one week at 32ºC in a shaker (120 rpm).

Table 1.

Composition of MN media used for ACPase induction

S. no.	Components	Final concentration (mg/L)
1	MgCl2	731
2	NaNO3	80
3	KCl	65
4	Ca(NO3)2.4H2O	288
5	Glucose	10,000
6	NaFeEDTA	8
7	KI	0.75
8	MnCl2.4H2O	6
9	C4H6O4Zn	2.65
10	H3BO3	1.5
11	CuCl2	0.13
12	Na2MoO4.2H2O	0.0024
13	KH2PO4 (Pi)	0.31a

^aConcentration is considered as a low Pi amount for induction of ACPase isoforms

Note: All compounds of MN media were dissolved in distilled water, and the pH was adjusted to 7 using 1 N NaOH and autoclaved for 15 min at 121 °C and 15 psi prior to use.

Chemicals and reagents

Sodium acetate trihydrate, α-naphthyl phosphate, and Fast Garnet GBC were procured from Sigma-Aldrich chemical Ltd. All the reagents were stored according to the manufacturer's instructions, and solutions were prepared immediately before use.

Preparation of sodium acetate buffer

1. Stock solution- To prepare 1 L of 0.1 M sodium acetate buffer, 13.6 g of sodium acetate trihydrate and 5.75 ml of acetic acid were added to 800 ml autoclaved MQ. The pH of the solution was maintained up to 4.5 with HCl, and the final volume was makeup to 1 L. Stock solutions were stored at 4 °C for one month.

2. Working solution- 0.05 M sodium acetate buffer (pH 4.5) was prepared by diluting the 0.1 M sodium acetate buffer. This solution can be stored at 4 °C for one month. 1 mM PMSF (protease inhibitor, final concentration) was added to the buffer before use. This buffer was used for ACPase isolation from the samples.

Sample preparation for ACPase isolation

All the materials used in the ACPase isolation were autoclaved and chilled (mortar pestle, buffer, 50 ml centrifugation tube, and 1.5 ml tube). Fungus incubated in MN media was filtered (Whatman No. 1) after 1 week, and mycelium was washed 10 times with autoclaved chilled MQ water and 3 times with ice-cold 1X PBS (100 mM) buffer to remove traces of media. The spent media (50 ml) was transferred to a chilled glass beaker and stored at 4 °C temperature for further use. Both mycelia and spent media were used for ACPase isolation.

Isolation of intracellular ACPase enzyme (IC)

1. One gm of fresh mycelial samples was crushed into fine powder using liquid nitrogen in a chilled mortar–pestle.

2. Powdered samples were transferred into a chilled 2 ml tube and re-suspended in 0.5 ml chilled 0.05 M sodium acetate buffer (containing 1 mM PMSF).

3. The tube was mixed using a vortex mixer (300 rpm) until homogeneous mixing was achieved in a 4 °C chamber (alternatively, tubes can be kept on ice for 1 min at a regular interval of 30 s to maintain a lower sample temperature).

4. The cell lysate containing a homogenous mixture of mycelia was centrifuged at 13,000×g (Eppendorf Centrifuge 5424 R) for 40 min at 4 °C.

5. The clear and transparent supernatant (I) was removed and used to identify intracellular ACPase isoforms.

   Note: If the transparent supernatant does not recover, STEP 4 can be repeated twice.

6. Aliquots of supernatant (crude proteins) in a 1.5 ml tube were stored at 4 °C for short-duration and at -80 °C with a flash-freezing in liq. N₂ for long duration and used for detection of intracellular ACPase isoform.

7. The remaining mycelial pellet was further washed three times with chilled sodium acetate buffer (0.05 M, pH 4.5) and used to isolate the cell-wall associated ACPase.

Isolation of cell wall-associated ACPase enzyme (CW)

1. The washed mycelial pellet was kept on ice to avoid heat-shock denaturation of protein during the sonication process.

2. Chilled sodium acetate buffer (0.05 M, pH 4.5) was added to the pellet (1:1)

3. Mycelial pellet was sonicated 3–5 times at 18 kHz for 30 s each with 60 s intervals (SONICS Vibra-Cell™Sonicator) or until the homogenization was achieved.

4. Homogenized cell-wall lysate was centrifuged at 13,000g for 40 min at 4 °C.

5. The transparent supernatant (II) was recovered and aliquoted in a 1.5 ml tube for storage (see step 6 of the previous section for the storage of protein samples).

6. This clear transparent supernatant (II) was used for the detection and expression of ACPase isoform associated with the cell wall in S. indica.

Isolation of extracellular/secretory ACPase (EC) from culture media by salt precipitation

1. Spent media was used for isolation of the secretory or extracellular ACPase.

2. A glass beaker containing spent media was kept in the ice to avoid exposure to heat or temperature fluctuations.

3. 11.67 g of solid ammonium sulphate was slowly added to the chilled media (50 ml) to obtain 40% saturated media. Slow and continuous stirring was maintained using a glass rod while adding the solid ammonium sulphate to chilled media.

4. After dissolving the ammonium sulphate, the beaker was kept at 4 °C temperature for 10 h.

5. After 10 h, media was transferred into 50 ml centrifugation tubes and centrifuged it at 13,000g for 40 min at 4 °C (Himac CR 22G High-Speed Refrigerated Centrifuge system).

6. The supernatant was discarded without disturbing the pellet at the bottom of the tube.

7. The tube was kept on ice, and the pellet was resuspended in 100 µl chilled 0.1 M sodium acetate buffer.

   Note: 0.1 M sodium acetate buffer was used to re-suspend the precipitated extracellular protein.

8. The protein sample was transferred into 1.5 ml of the tube and stored at − 80 °C for subsequent use of extracellular ACPase isoform detection and expression in S. indica.

Note:

• Maintain 4 °C temperature during ammonium sulphate precipitation and avoid frothing while dissolving the ammonium sulphate.

• Magnetic stirrer not recommended in step 3; it may generate heat and impact the recovery.

• Protein isolations were performed in 5 replicates.

In-gel assay protocol for ACPase

Native-PAGE acrylamide gel was used for the identification and expression of ACPase isoforms in all prepared samples (Barka 1961; MacIntyre 1971). All the accessories required for gel electrophoresis were treated with 70% ethanol for 5 min and washed with autoclaved MQ before use.

Reagent preparation

10X running buffer (pH 8.3): 30.285 g Tris (25 mM) and 144.1344 g (192 mM) glycine were dissolved in 500 ml MQ, and pH (8.3) was maintained before the final volume was adjusted to 1 L. The 1X running buffer (chilled) was used for electrophoresis.

30% acrylamide solution: 29.22 g of acrylamide and 1 g of bis-acrylamide were added in 80 ml autoclaved MQ, and volume was adjusted up to 100 ml. The solution was filtered and stored at 4 °C at least for 1 month.

0.375 M Tris–HCl (pH 8.8): 4.54 g of Tris-base was added in 80 ml MQ, and pH was adjusted to 8.8 before the final volume was adjusted to 100 ml.

Sample buffer (2x): 100 ml of sample buffer was prepared by adding 0.75713 g Tris–HCl (62.5 mM, pH 6.8), 25% glycerol (final concentration), and 1% bromophenol blue (final concentration) in MQ.

Native-PAGE staining solution for in-gel assay activity: 0.1% α-naphthyl phosphate and 0.1% Fast Garnet GBC were dissolved in chilled 0.1 M sodium acetate (pH 4.5) and filtered. 5 mM MgCl₂ was added in a filtered solution. The fresh staining solution was stored at 4 °C in the dark until use (MacIntyre 1971; Straker and Mitchell 1986).

Note: Use freshly prepared staining solution. Add MgCl₂ after filtration of α-naphthyl phosphate and Fast Garnet GBC solution to avoid precipitation. Precipitation reduces the staining quality of the gel. The absence of MgCl₂ in the staining solution reduces the contrast band formation on the gel (Fig. 1B).

Fig. 1.

Native-PAGE for ACPase enzyme and associated issues in its assay. A Native-PAGE shows stacked isoforms due to a problem in resolving of bands; B low visualization of the band in the absence of MgCl₂

De-staining solution: 0.1% acetic acid (v/v) was used for de-staining and to store the native-PAGE gel.

Composition of native-PAGE gel

The composition of native-PAGE is summarized in Tables 2 and 3.

Table 2.

Composition of 8% resolving gel for native-PAGE

S. no.	Components	Volume (ml) used for preparing the 8% resolving gel
1	8% acrylamide/bis-acrylamide	2.6 ml of 30% acrylamide/bis-acrylamide solution
2	0.375 M Tris–HCl, pH 8.8	7.29
3	10% ammonium persulfate	0.1
4	TEMED	0.01

APS and TEMED were added right before use

Table 3.

Composition of 5% stacking gel for native-PAGE

S. no.	Components	Volume (ml) used for preparing the 5% stacking gel
1	4% acrylamide/bis-acrylamide	0.67 ml of 30% acrylamide/bis-acrylamide solution
2	0.375 M Tris–HCl, pH 8.8	4.275
3	10% ammonium persulfate	0.05
4	TEMED	0.005

APS and TEMED were added right before use

Assembly of cassettes

1. Mini-electrophoresis system (BIO-RAD) was used for native-PAGE.

2. 1.5 mm spacer plates, short plates, casting frame, and 1.5 mm comb were cleaned with 70% ethanol.

3. The spacer plate and the short plate were fixed on the casting frame.

4. The casting frame with glass plates was fixed on the casting stand with the help of clamps.

Note: Assemble the cassettes as per instructions for other designs.

Gel preparation

1. An 8% (10 ml) resolving gel solution was prepared in a 15 ml polypropylene tube according to Table 2.

2. The gap between the spacer plate and short plate was quickly filled with the resolving gel solution using a pipette until the gel solution reached 1.5 cm fewer to top edge of short plate. One ml of isopropanol was added to the gel surface to form a uniform layer of polymerized gel.

3. The gel was incubated for 20–30 min at room temperature for complete polymerization.

4. Isopropanol was removed from the top of the resolving gel by tilting the gel aside.

5. The upper layer of the gel was washed with MQ (2–3 times).

6. A 5% stacking gel solution was prepared into a 15 ml tube using components mentioned in Table 2.

7. The stacking gel was quickly filled above the resolving gel using the pipette.

8. The comb was carefully placed between the glass plate spaces.

   Note: Be careful not to introduce air bubbles. Avoid the development of air bubbles while fixing the comb.

9. The gel was left for the next 20 min for polymerization.

Sample preparation: 5 µg for IC and CW samples and 3 µg of EC sample were mixed to the sample buffer (1X) at a final dilution. Note: No heating is required in the sample preparation. Samples were centrifuged (5000 g for 1 min) at 4 °C and stored at 4 °C until loading.

Gel running conditions

1. The polymerized gels were removed from casting frame and fixed in the electrophoresis cell. Cathode chamber of the electrophoresis cell was filled with pre-chilled electrophoresis running buffer (1X).

2. The comb was removed from the gel after filling the anode chamber of the electrophoresis unit with pre-chilled electrophoresis running buffer (1X).

3. Electrophoresis apparatus was pre-run to equilibrate the gel at 70 V for 1 h at 4 °C.

4. After 1 h, the bubbles were removed carefully and the prepared samples were loaded in a well using micro-syringe (Hamilton).

5. The entire electrophoresis cell was kept at 4 °C to reduce the heating of electrophoresis cell during the electrophoresis. The gel was run at 70 V at 4 °C for 8 h.

Note:

• The sample should be run for at least 4 more hours, after reaching the dye at the bottom of the gel for better separation of isoforms on the gel (not following this step will results into the improper separation of isoforms, Fig. 1A).

• All the reagents and apparatus should be pre-chilled before use.

In-gel staining procedure: Gel staining was done using the (MacIntyre 1971; Straker and Mitchell 1986) method with few modifications. The gel was removed from the plates, placed into a staining box, and incubated in chilled 0.1 M sodium acetate buffer for an hour. The sodium acetate buffer was removed and gel was incubated in staining solution for 3–5 h at room temperature in the dark on a rocker (15 rpm). The staining solutions was discarded after developing dark red or brown band on gel. The gel was washed carefully with MQ (5–6 times) with 5 min intervals between each round of washing. The image of the stained gel was taken using ChemiImager™ 4400 (Alpha Innotech). The gel was stored in 5% ice-cold acetic acid at room temperature (alternatively, the stained gel may be wrapped in cling film and dried).

Note: Gel staining must be carried out in the dark at room temperature. Light exposure can inhibit the staining process.

Results

Identifying the enzyme isoforms is a tedious task when the cell produces isoforms with a different location within the cell. To identify the ACPAse isoform in the filamentous fungi S. indica, the native protein isolated from fungus was separated by electrophoresis on 8% native gel and activity assay was performed. Red/brown colored bands appeared on the gel, indicating ACPase activity (Fig. 2). A clear band of activity was visualized for the corresponding ACPase isoform separated in the gel, indicating that the protocol successfully identified intracellular, extracellular, and cell wall-associated ACPase isoforms in S. indica. Duration of electrophoresis is an essential factor in resolving enzymes on the gel to visualize clear and separated bands of the isoforms. An issue of separation of isoforms was observed in the native gel (Fig. 1A), where isoforms were stacked and were not separated well. Here, our crucial and effortless modification resolved this problem by increasing electrophoresis time (please see step 5 and note in the gel running condition section).

Fig. 2.

Native-PAGE profiles of the ACPase isoforms and its expression in S. indica. 5 μg native protein of intracellular (IC), cell wall-associated (CW), and 3 μg of extracellular (EC) ACPase was separated by Native-PAGE acrylamide electrophoresis, and the gel was stained with 0.1% α-naphthyl phosphate and 0.1% Fast Garnet GBC. Two isoforms of ACPase were expressed intracellularly and one isoform in both cell wall and extracellularly (indicated by black arrow)

Several modifications are necessary to enhance the quality of band visualization and contrast in the gel band. We observed that the addition of α-naphthyl phosphate and fast garnet GBC with MgCl₂ to staining solution results in the formation of precipitates which reduces the quality of gel for imaging. However, if a simple modification was applied where α-naphthyl phosphate and fast garnet GBC were dissolved in a buffer first and after filtering this solution, adding MgCl₂ to it results in reduced precipitation, thereby enhancing gel quality. Further, no addition of MgCl₂ results in low band quality (Fig. 1B; please see note in the Native-PAGE staining solution for in-gel assay activity section).

The major problem associated with this enzyme is that it is a repressible ACPase which means if a trace amount of the Pi will be available in the media, it represses the expression of ACPase. To resolve this problem, we modified the culture media and conditions of S. indica to induce and isolate all ACPase isoforms in their native form for isoforms identification (please see “Fungal culture preparation” and “Change of media for ACPase induction and secretion”). The new modified minimal media was used to de-repress the ACPase gene and its expression (Table 1). This protocol uses as small as 5 μg (IC and CW) and 3 μg (EC) of native protein for in-gel native-PAGE assay to identify ACPase isoforms in S. indica. Our analysis suggests that S. indica has two isoforms for ACPase, and both isoforms were expressed in the phosphate starvation condition. Out of the two isoforms, one is secretory. We also detected it on the cell-wall fraction and media fraction proteins through native PAGE in-gel assay. At the same time, another isoform was also found intracellular in localization as we have not detected this isoform in the cell wall and media fraction (Fig. 2).

In the present protocol, single culture was used to process all isoforms of ACPases unlike the earlier report in endomycorrhizal fungi (Straker and Mitchell 1986), where fungi were incubated for 13-day-long period for induction of ACPase, multiple steps were used for extracellular ACPase isolation and also not mentioned the starting amount of mycelia (gm) required for ACPase isolation. Aleksieva et al. (2003) isolated IC and EC ACPase, but no standard protocol was described for isoforms detection and expression analysis (Aleksieva et al. 2003).

This protocol has an advantage over the others as one can isolate all forms of ACPase from a single fungal culture. For this purpose, fungus culture was incubated in MN media for one week to induce ACPase isoforms. Isolation of extracellular ACPase was not easy since it was available in the medium (highly diluted), and its direct loading onto native-PAGE was impossible. In this protocol, only two steps are required to isolate EC ACPase. The extracellular ACPase enzyme was obtained after 40% ammonium sulfate precipitation and dialysis, increasing purification about 26 times higher (Table 4), and there was no need to concentrate the protein as reported earlier (Straker and Mitchell 1986). The protocol was designed for efficient isolation and identification of ACPase isoforms in their native form.

Table 4.

Summary of the purification of extracellular ACPase

S. no.	Purification step	Total protein (mg/ml)	Activity (unit)	Specific (Unit/mg)	Purification fold
1	At media stage	0.034926	0.000150273	0.00430256	1
2	After precipitation and dialysis	1.7494387	0.200045177	0.114351775	26.57761503

Discussion

The present study aimed to establish the optimal protocols for analyzing ACPase isoforms from filamentous fungus S. indica. The detection of ACPase isoforms by the in-gel assay technique depends on the induction and expression in the growth media. The standardization of repression and de-repression conditions for Pi responsive genes during the growth has not been established in most root colonizing arbuscular mycorrhizal fungi (AMF) fungi.

The AMF association improves the acquisition of Pi in the host plant from the soil by extending its hyphae. Secretory ACPase plays a crucial role in this process and is involved in P-solubilization to increase the amount of Pi in the rhizosphere soil. The detection and identification of ACPase isoforms activated under P-deficiency conditions have not been well studied in AMF because of the unavailability of their axenic culture. Several investigators reported intracellular ACPase isoforms. However, extracellular or secretory ACPase has been demonstrated only in a few AMF (Sato et al. 2015) because of its symbiotic association to the host and, therefore, identifying secretory/extracellular ACPase in such fungi is not easy. Few studies established a monoxenic culture to isolate secretory ACPase from mycorrhizal fungi and perform SDS-PAGE for ACPase activity (Sato et al. 2015). This time-consuming process includes multiple modified culture conditions to perform activity assay only for secretory ACPase.

In comparison, this native-PAGE activity assay was more efficient in identifying isoforms of enzymes. Our results indicate that axenically, the AM-like fungus S. indica can express ACPase isoforms under a P-deficient environment. The present protocol efficiently identifies ACPase isoforms expressed in a P-deficient environment from a single culture. Moreover, the protocol is simple, easy, and cost-effective to follow. All isoforms of ACPase were identified within a single sample. The protocol is reliable and consistent and operates effectively even in the axenic and monoxenic cultures. This protocol is also valuable for identifying secretory ACPase in culture media, promoting P-solubilization in rhizospheric soil.

Author contributions

ASK: designing, protocol and experiment standardization, formal analysis, visualization, data analysis, literature survey and manuscript writing. MK: conception, data analyzing and manuscript editing.

Funding

ASK is thankful to the UGC for providing Senior Research Fellowship to support her PhD research work. MK is thankful to the CSIR-Indian Institute of Toxicology Research, Lucknow, India for providing financial support (MLP002).

Availability of data and material (data transparency)

All data related to this work will be available on request to the corresponding author.

Code availability (software application or custom code)

Not applicable.

Declarations

Conflict of interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence work reported in this paper.

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All authors have seen, read, agreed and given consent for the communication and publication of this manuscript. The CSIR-IITR communication number for this manuscript is 3752.