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Full Research Article

Biochemical Alterations in Sodium Azide Treated Vigna radiata during Seed Germination

A
Ajit Sopan Masurkar1,2
A
Aniket Pramod Phadtare1
K
Kumari Monika1
S
S. Indu Kumari1,*
1Division of Applied Biology, CSIR-Indian Institute of Chemical Technology, Tarnaka, Hyderabad-500 007, Telangana, India.
2Academy of Scientific and Innovative Research (AcSIR), Ghaziabad-201 002, Uttar Pradesh, India.

ABSTRACT

Background: Sodium azide is a threat to the ecosystem and agriculture due to its high toxicity and increasing production. It is known to reduce seed germination in many plants with unknown mechanism of action.

Methods: Seeds of Vigna radiata were treated with different concentrations of sodium azide using 0.1 M phosphate buffer pH 3.2 as a vehicle and control. Using seed germination assay data, 3 mM sodium azide was selected for further study which includes estimation of proteins, reducing sugar, α-amylase gene expression and activity, antioxidant activity and mineral analysis.

Result: Our findings show that sodium azide at 2 mM concentration and above reduced Vigna radiata seed germination significantly. The treatment of Vigna radiata seeds with 3 mM sodium azide for 3 h showed physiological changes in germination affecting protein and reducing sugar content. These changes can be related to the low α-amylase activity in seeds treated with 3 mM sodium azide. Compared to control, treated seeds showed significant reduction in α-amylase activity per seed by 29, 40 and 41% at 30oC incubation for 24, 48 and 72 h respectively. This reduction in enzyme activity is supported by the inhibition of α-amylase gene expression. 3 mM sodium azide treatment causes reduction in antioxidant defense of germinating seeds by reducing the total phenolic content and DPPH quenching activity compared to control and vechile control treated seeds. Hence, it can be concluded that α-amylase inhibition at 3 mM sodium azide, along with oxidative stress, leads to germination failure.

INTRODUCTION

Seed germination, essential for plant development, involves changes in transcript, protein and hormone levels (Bewley et al., 1997). Plant hormones (gibberellic acid, abscisic acid) and enzymes like α-amylase regulate this process (Urbanova et al., 2016). α-amylase breaks down starch into sugars for energy (Kaneko et al., 2002). In Jatropha curcas, carbohydrate levels decrease during germination, indicating high utilization (Lopes et al., 2013). Free radical production is a stress factor, emphasizing the role of antioxidants and enzymes in successful germination (Bailly, 2004).
       
Various organic and inorganic substances released into the environment disrupt plant life cycles and harm the ecosystem. Sodium azide (NaN3), a highly toxic compound with an unclear mode of action, has seen increased use over the past 30 years in airbags, pest control, fumigation (Tat et al., 2021) and as a mutagen (Jahan et al., 2020). At the cellular level, SA affects cell functioning by inhibiting mitochondrial cytochrome C oxidase and catalase (Keilin and Hartree, 1934; Stannard and horecker, 1948). Several studies on SA show that it inhibits seed germination in tomato (Adamu and Aliya, 2007), Eruca sativa (Khan et al., 2009), mungbean (Ali et al., 2024) and rice (Awan et al., 1980) which badly affect their growth and survival. It’s mode of action preventing seed germination is unknown. In this study the influence of SA upon the levels of macromolecules, gene expression and the activity of α-amylase enzyme, antioxidant defence and changes in mineral content during seed germination were recorded. These findings suggest that SA causes oxidative stress and reduces α-amylase activity, which resulted in failure of seed germination.

MATERIALS AND METHODS

Plant material
 
The Vigna radiata seeds (IPM-2-14) were procured from Indian Institute of Pulse Research Kanpur, India. 
       
The entire experiment was conducted in 2022-2024 at the Applied Biology Division, CSIR- Indian Institute of Chemical Technology, Hyderabad, Telangana.
 
Germination treatment and Seed germination assay
 
Vigna radiata (IPM-2-14) seeds were rinsed with autoclaved water, surface sterilized with 70% ethanol for 2 min and rinsed thrice to remove ethanol. Seeds were soaked in autoclaved water for 14 h at 25oC to initiate germination, then treated for 3 h at room temperature on a shaker with SA (1-10 mM in 0.1 M phosphate buffer, pH 3.2), water (control), or buffer (vehicle control). Treated seeds were washed, placed on moist filter paper in sterile petriplates and incubated at 30oC. Germination was recorded daily for 6 days. Per cent germination was calculated every 48 h and the SA concentration causing ~50% germination on day 4 was used for toxicity analysis.
 
Estimation of reducing sugars and proteins
 
Crude extract preparation was as per Sottirattanapan et al. (2017). Crude extract reducing sugars and protein content were estimated by DNS (3, 5-dinitrosalicylic acid) and Bradford methods respectively.
 
Qualitative assay for α-amylase
 
The Vigna radiata seeds used in the study were treated with SA, VC and control. Embryo less half seeds were tested to analyze the qualitative reduction in α-amylase activity on treating with SA (Liu et al., 2018). The α-amylase activity which hydrolyzed the starch is represented by the colorless zone.
 
Quantitative assay for α-amylase
 
The treated, control and VC Vigna radiata seeds were collected after incubation at 30oC for 24, 48 and 72 h. Five seeds from each treatment were homogenized using glass mortar and pestle in pre-chilled 10 ml of 0.1 M sodium acetate buffer pH 5.6, kept at 4oC for 10 min then centrifuged at 10,000 rpm for 10 min at 4oC. The supernatant was used for quantification of α-amylase activity. Quantification of α-amylase was done by DNS method of Miller (1959). Maltose was used as a standard to estimate the reducing sugars released due to starch degradation by α-amylase enzyme in crude extract using a spectrophotometer at 540 nm. One unit of α-amylase activity was defined as the quantity of enzyme required to release 1 µM of maltose per minute.
 
Gene expression of α-amylase
 
RNA isolation
 
Seeds treated with 3 mM SA, VC and control were transferred to autoclaved petriplates layered with moist filter paper and kept at 30oC in oven followed by RNA isolation at intervals of 0, 24, 48 and 72 h after treatment by method of Meng and Feldman (2010).  The RNA pellet was dissolved in 50 μl DEPC-treated, autoclaved ddH2O and stored at -80oC.
 
cDNA synthesis and qR-PCR
 
Total extracted RNA was quantified with Nanodrop (Thermo Scientific-NanoDrop™ 2000c Spectrophotometer). Further 1 µg of total RNA was used to prepare cDNA using TaKaRa Prime Script™ 1st strand cDNA Synthesis Kit (Cat. #6110A) according to the manufacturers protocol. Quantitative Real Time PCR (q-RT PCR) was performed using TaKaRa TB Green® Premix Ex Taq™ II (Tli RNaseH Plus) (Cat. #RR820A) according to kit specified standard protocol. Comparative CT Value method (2-ΔΔCt) was used for the quantification of Gene expressions (Balaji et al., 2016).

Free radical scavenging assay and total phenolic content
 
Extract was prepared according to method of Wei et al. (2019) to estimate free radical scavenging activity and total phenolic content. To estimate free radical scavenging activity 2 ml of 0.2 mM DPPH in ethanol was mixed with 0.5 ml of extract. This mixture was incubated in dark for 30 min at room temperature. Absorbance was measured at 570 nm using spectrophotometer. Total phenolic content was estimated according to method of Xu and Chang (2007). The total phenolic content was expressed as gallic acid equivalents (mg of GAE/g sample) through the calibration curve of gallic acid.
 
Mineral analysis
 
Seeds of green gram were treated as mentioned above with 3 mM SA, Control and VC. Seeds were thoroughly washed with autoclaved distilled water and incubated at 30oC for 24 and 48 h. After incubation samples were prepared according to Alkaltham et al. (2020) to analyse calcium (Ca), potassium (K), sodium (Na), iron (Fe),  copper (Cu), zinc (Zn), magnesium (Mg) and manganese (Mn) using ICP-OES (CSIR-IICT, Hyderabad, India).
 
Statistical analysis
 
Two-way ANOVA was performed for obtaining statistical significance. Graph-pad prism software version 8.0.2 was used for all statistical tests and plotting the graphs of gene expression studies. One-way analysis of variance (ANOVA) followed by Tukey test was performed by using instat 3 software for analysis of other data. All tests were performed in at least three replicates and the data are expressed as means ± standard deviation (SD). Significant difference compared to control for same time period are indicated by *(P<0.05), **(P<0.01) and ***(P<0.001).

RESULTS AND DISCUSSION

Seed germination assay
 
On 4 day, per cent seed germination data obtained was same as control and VC for 1 mM of SA. Per cent germination was reduced up to 82%, 38%, 23%, 00% for 3 h treatment of 2, 3, 4 and 5 mM SA respectively (Fig 1). These results revealed that, SA at concentration of 2 mM and above significantly affected the germination of Vigna radiata seeds. SA at the concentration of 5 mM and above showed complete reduction in germination of Vigna radiata seeds.

Fig 1: Effect of sodium azide on green gram seed germination.


 
Biomolecular assay
 
Reducing sugars in SA, VC and control-treated seeds were quantified using the 3, 5-Dinitrosalicylic acid method Miller (1959). The control and VC showed a significant increase in reducing sugars At 24, 48 and 72 h , while SA-treated seeds did not (Fig 2A). Protein content, analyzed by the Bradford method showed no significant difference immediately after treatment. At 24 and 48 h, control and VC had reduced protein, while SA-treated seeds showed no change (Fig 2B).

Fig 2A: Effect of sodium azide treatment on reducing sugar content of germinating seeds.



Fig 2B: Effect of sodium azide treatment on protein content of germinating seeds.



α-amylase gene expression study
 
Gene expression analysis at 0 h condition of 3 mM SA in acidic phosphate buffer (pH-3.2) treated Vigna radiata seeds showed similar expression of α-amylase as in control and VC treated seeds. The expression of α-amylase down regulated in 3 mM treated seeds compared to the control and VC under 24 h condition. However, an up regulated expression of α-amylase was observed in VC, compared to the control samples at 48 h. The expression of α-amylase was found to decline in seeds treated with 3 mM SA with the increasing incubation time (Fig 3).

Fig 3: Differential expression of α-amylase.


 
α-amylase activity
 
The Vigna radiata seeds treated with 3 mM SA for 3 h, incubated for 24, 48 and 72 h to evaluate effect of SA on the α-amylase activity.
 
Quantitative assay
 
Quantitative assay for α-amylase reveals that 3 mM SA treatment significantly decreased α-amylase activity as compared to control and VC. Immediately after treatment no significant differences were observed between α-amylase activity of control, VC and treated seeds. As compared to the control, seeds treated with 3 mM SA showed reduction in α-amylase activity by 29%, 40% and 41% at 24, 48 and 72 h incubation period at 30oC respectively. As compared to the VC, seeds treated with 3 mM SA showed reduction in α-amylase activity by 23%, 52% and 34% at 24, 48 and 72 h incubation period at 30oC respectively (Fig 4A).

Fig 4A: Effect of Control, vehicle control and sodium azide on á-amylase activity in germinating seeds of green gram.


 
Qualitative assay
 
Qualitative assay was performed to assess the effect of SA on the enzyme activity after incubation of 48 h. The control and VC seeds showed greater more transparent area around them as compared to seeds treated with SA (Fig 4B).

Fig 4B: Effect of Control, vehicle control and sodium azide on á-amylase activity in germinating seeds of green gram.


 
Free radical scavenging activity and total phenolic content
 
% DPPH scavenging activity in 3 mM treated seeds was significantly less as compared to control and VC treated seeds after 24, 48 and 72 h of incubation after post treatment (Table 1). Total phenolic content of control and VC treated seeds increased after 72 h of incubation but comparatively less increase was observed in 3 mM treated seeds (Table 1).

Table 1: % DPPH scavenging activity and total phenolic content (TPC) of 3 mM SA ( sodium azide), control and VC ( vehicle control) treated seeds at different time incubation.


 
Mineral analysis
 
Seeds treated with 3 mM SA, control and VC as mentioned above were tested for mineral changes in such as Ca, Na, K, Fe, Cu, Zn, Mg and Mn during seed germination. Out of these eight tested minerals only Ca, Na and Fe showed statistically significant differences for the mentioned treatments. SA treatment showed rise in Ca content of treated seeds at 0 h of treatment compared to VC and control treated seeds while after 48 h of SA treatment Ca content in both VC and SA treated seeds decreased compared to control which was statistically significant (Table 2). An increase in Fe content after 24 h of treatment in SA treated seeds compared to control and VC treated seeds and with normal levels at 48 h of incubation was noted (Table 2). Na content in SA treated seeds was higher compared to both control and VC treated seeds at 0 and 48 h of incubation with a statistically significant rise as compared to control (Table 2).

Table 2: Mineral content (mg/100 g) of 3 mM SA (sodium azide), control and VC ( vehicle control) treated seeds at different time incubation.


       
Seed germination assay
 
Seed germination inhibition by SA at concentrations ≥ 1 mM had been reported in Lycopersicon esculentum (Adamu and Aliya, 2007) and Eruca sativa (Khan et al., 2009). Our study on Vigna radiata showed a concentration-dependent decline in germination with increasing SA levels. Germination dropped significantly at 2 mM and was completely inhibited (0%) at 5 mM. These results align with the previous findings in Cajanus cajan (Chaudhary et al., 2021), confirming SA’s strong inhibitory effect.
 
Biomolecular changes
 
Seeds treated with 3 mM SA showed declined reducing sugar content, indicating impaired starch hydrolysis. Starch breaks down into glucose and maltose, providing energy for germination (Kaneko et al., 2002). Normally, germination involves starch breakdown for energy and protein degradation into amino acids (Palmiano and Juliano, 1972). In control and VC seeds, protein content decreased over time, while SA-treated seeds showed no significant change, suggesting inhibited protein reserve use. Similar trends were noted in grassland species, with rising sugar and declining protein levels during germination (Zhao et al., 2018). These effects may result from reduced α-amylase activity, crucial for starch breakdown and germination.
 
α-amylase activity
 
SA-treated seeds showed reduced gene expression and α-amylase activity, while VC seeds showed increased expression at 48 h. Combined SA and VC treatment confirmed SA’s inhibitory effect. α-amylase is vital for breaking down reserves during germination (Damaris et al., 2019), matching our observation as small transparent zones around the treated seeds in our study. Control and VC seeds showed rising enzyme activity unlike in SA-treated seeds. Similar reductions were observed under NaCl stress in rice (Liu et al., 2018) and during germination in rice and peas (Murata et al., 1968; Juliano and Varner, 1969).  Quantitative data supported qualitative results. In Pisum sativum, cadmium reduced germination via low amylase activity (Chugh and Sawhveey, 1996). Likewise, our study with SA treatment had led to high protein, low sugar, limited energy with failed germination. These findings match Ashraf et al. (2002), who linked reduced α-amylase activity to poor germination under salinity.
 
Antioxidant activity
 
The imbalance between excessive generation of reactive species and antioxidant defence leads to oxidative stress which disturbs the redox homeostasis, damages membrane lipids, proteins and nucleic acids, killing the plant cell (Jain and Shakkarpude, 2024). ROS interacts with various enzymes and inhibits them in turn affecting the physiology of the plant. Our study shows parallel reduction in both antioxidant defence and α-amylase activity due to SA stress which is similar to Zang et al. (2008). Our results show that SA treatment affected seed germination in Vigna radiata by reducing free radical scavenging activity and total phenolic content which act as protective measures of the cell during oxidative stress.
 
Mineral analysis
 
SA treatment increased Ca2+ levels by 44%, suggesting an oxidative stress. Pang and Wang (2008) noted that ionic stress leads to ROS accumulation, countered by antioxidants. An antioxidant failure damages the plant growth. Elevated Na in SA-treated seeds at 0, 24 and 48 h indicates failed germination. Salt stress can increase ABA, reduce gibberellic acid, hinder water uptake and cause toxicity, all of which impair seed germination and development (Nikolić et al., 2023). Excessive iron triggers oxidative stress and reduced growth in Chlorella vulgaris (Estevez et al., 2001). Our data shows increased Fe in SA-treated seeds after 24 h. Calcium, Sodium and Iron accumulation may cause ROS buildup, damaging seeds due to reduced oxidative defence.

CONCLUSION

Our study concludes that at 3 mM SA treatment seed germination in Vigna radiata and α-amylase enzyme activity was reduced, which in turn resulted in failure of starch and protein reserve utilization for their development. This reduction in enzyme activity is related to its low gene expression which suggests that SA might interfere at transcriptional level of α-amylase gene expression. Further due to SA treatment elevation in Ca, Fe and Na levels were recorded which could be responsible in causing oxidative damage. We conclude, hampered biochemistry of SA treated seeds could be a mixed response of elevation in Ca, Fe and Na, oxidative stress, reduction in antioxidant defence and inhibition of α-amylase enzyme.

ACKNOWLEDGEMENT

We thank the Director, CSIR-Indian Institute of Chemical Technology (IICT) (Ms. No. IICT/Pubs./2022/290.) For providing all the required facilities to carry out the project work. Masurkar Ajit Sopan (SRF) and Phadtare Aniket Pramod (JRF) are grateful to the University Grant Commission (UGC) for the award of the fellowship.

CONFLICT OF INTEREST

The authors declare that there are no conflicts of interest related to this research.

REFERENCES


  1. Adamu, A.K. and Aliyu, H. (2007). Morphogical effects of sodium azide on tomato (Lycopersiconesculentum Mill). Science World Journal. 2(4): 9-12. doi: 10.4314/swj.v2i4.51755.

  2. Ali Basid, S.M.D., Konjengbam, S.N., Ahmad, F., Sanasam, S. and Kumawat, R. (2024). Ascertaining lethal dose 50 (LD50) and simultaneous effect of ethyl methane sulphonate (EMS) and sodium azide (SA) on seedling characters in mungbean genotypes 'Pusa 1031' and 'Pusa 1431'. Legume Research. 47(11): 1929-1935. doi: 10.18805/LR-5255.

  3. Alkaltham, M.S., Salamatullah, A.M., Özcan, M.M., Uslu, N. and Hayat, K. (2020). The effects of germination and heating on bioactive properties, phenolic compounds and mineral contents of green gram seeds. LWT. 134: 110106.

  4. Ashraf, M.Y., Sarwar, G., Ashraf, M., Afaf, R. and Sattar, A. (2002). Salinity induced changes in α-amylase activity during germination and early cotton seedling growth. Biologia Plantarum. 45(4): 589-591.

  5. Awan, M.A., Konzak, C.F., Rutger, J.N. and Nilan, R.A. (1980). Mutagenic effects of sodium azide in rice 1. Crop Science. 20(5): 663-668.

  6. Bailly, C. (2004). Active oxygen species and antioxidants in seed biology. Seed Science Research. 14(2): 93-107.

  7. Balaji, S.A., Udupa, N., Chamallamudi, M.R., Gupta, V. and Rangarajan, A. (2016). Role of the drug transporter ABCC3 in breast cancer chemoresistance. PloS one. 11(5): e0155013.

  8. Bewley, J.D. (1997). Seed germination and dormancy. The plant cell. 9(7): 1055.

  9. Chaudhary, L., Sharma, R. and Kumar, M. (2021). Estimation of LD50 and effect of sodium azide on germination and seedling parameters of different cultivars of Cajanus cajan (L.) Millspaugh. Toxicology and Environmental Health Sciences. 13(3): 279-285.

  10. Chugh, L.K. and Sawhney, S.K. (1996). Effect of cadmium on germination, amylases and rate of respiration of germinating pea seeds. Environmental Pollution. 92(1): 1-5.

  11. Damaris, R.N., Lin, Z., Yang, P. and He, D. (2019). The rice alpha-amylase, conserved regulator of seed maturation and germination. International Journal of Molecular Sciences. 20(2): 450.

  12. Estevez, M.S., Malanga, G. and Puntarulo, S. (2001). Iron-dependent oxidative stress in Chlorella vulgaris. Plant Science. 161(1): 9-17.

  13. Jahan, R., Malik, S., Ansari, B.S. and Khan, S. (2020). Evaluation of optimal doses for gamma rays and sodium azide in linseed genotypes. Agricultural Science Digest. 41(Special Issue): 207- 210. doi: 10.18805/ag.D-5165.

  14. Jain, A. and Shakkarpude, J. (2024). Oxidative stress: A biomarker for animal health and production: A review. Indian Journal of Animal Research. 58(1): 01-12. doi: 10.18805/IJAR.B-5300.

  15. Juliano, B.O. and Varner, J.E. (1969). Enzymic degradation of starch granules in the cotyledons of germinating peas. Plant Physiology. 44(6): 886-892.

  16. Kaneko, M., Itoh, H., Ueguchi-Tanaka, M., Ashikari, M. and Matsuoka, M. (2002). The α-amylase induction in endosperm during rice seed germination is caused by gibberellin synthesized in epithelium. Plant Physiology. 128(4): 1264-1270.

  17. Keilin, D. and Hartree, E.F. (1934). Inhibitors of catalase reaction. Nature. 134(3398): 933-933.

  18. Khan, S. and Al-Qurainy, F. (2009). Mutagenic effect of sodium azide on seed germination of Eruca sativa (L.). Australian Journal of Basic and Applied Sciences. 3(4): 3081-3087.

  19. Liu, L., Xia, W., Li, H., Zeng, H., Wei, B., Han, S. and Yin, C. (2018). Salinity inhibits rice seed germination by reducing α-amylase activity via decreased bioactive gibberellin content. Frontiers in Plant Science. 9: 275.

  20. Lopes, L.D.S., Gallão, M.I. and Bertini, C.H.C.D.M. (2013). Mobilisation of reserves during germination of Jatropha seeds. Revista Ciência Agronômica. 44: 371-378.

  21. Meng, L. and Feldman, L. (2010). A rapid TRIzol based two step method for DNA free RNA extraction from Arabidopsis siliques and dry seeds Weinheim: WILEY VCH Verlag. 5(2): 183-186.

  22. Miller, G.L. (1959). Use of dinitrosalicylic acid reagent for determination of reducing sugar. Analytical Chemistry. 31(3): 426-428.

  23. Murata, T., Akazawa, T. and Fukuchi, S. (1968). Enzymic mechanism of starch breakdown in germinating rice seeds I. An analytical study. Plant Physiology. 43(12): 1899-1905.

  24. Nikolić, N., Ghirardelli, A., Schiavon, M. and Masin, R. (2023). Effects of the salinity-temperature interaction on seed germination and early seedling development: a comparative study of crop and weed species. BMC Plant Biology. 23(1): 446.

  25. Palmiano, E.P. and Juliano, B.O. (1972). Biochemical changes in the rice grain during germination. Plant Physiology. 49(5): 751-756.

  26. Pang, C.H. and Wang, B.S. (2008). Oxidative stress and salt tolerance in plants. Progress in Botany. 231-245.

  27. Sottirattanapan, P., Nantachai, K., Daduang, S., Funahashi, T. and Yamada, M. (2017). Purification and characterization of amylase from roots of Paederiafoetida Linn. Biocatalysis and Agricultural Biotechnology. 10: 329-335.

  28. Stannard, J.N. and Horecker, B.L. (1948). The in vitro inhibition of cytochrome oxidase by azide and cyanide. J. Biol Chem. 172(2): 599-608.

  29. Tat, J., Heskett, K., Satomi, S., Pilz, R.B., Golomb, B.A. and Boss, G.R. (2021). Sodium azide poisoning: A narrative review. Clinical Toxicology. 59(8): 683-697.

  30. Urbanova, T. and Leubner Metzger, G. (2016). Gibberellins and seed germination. Annual Plant Reviews. 49: 253-284.

  31. Wei, Y., Wang, X., Shao, X., Xu, F. and Wang, H. (2019). Sucrose treatment of mung bean seeds results in increased vitamin C, total phenolics and antioxidant activity in mung bean sprouts. Food Science and Nutrition. 7(12): 4037-4044.

  32. Xu, B.J. and Chang, S.K.C. (2007). A comparative study on phenolic profiles and antioxidant activities of legumes as affected by extraction solvents. Journal of Food Science. 72(2): S159-S166.

  33. Zhang, H., Hu, L.Y., Hu, K.D., He, Y.D., Wang, S.H. and Luo, J.P. (2008). Hydrogen sulfide promotes wheat seed germination and alleviates oxidative damage against copper stress. Journal of Integrative Plant Biology. 50(12): 1518-1529.

  34. Zhao, M., Zhang, H., Yan, H., Qiu, L. and Baskin, C.C. (2018). Mobilization and role of starch, protein and fat reserves during seed germination of six wild grassland species. Frontiers in Plant Science. 9: 234.
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    Full Research Article

    Biochemical Alterations in Sodium Azide Treated Vigna radiata during Seed Germination

    A
    Ajit Sopan Masurkar1,2
    A
    Aniket Pramod Phadtare1
    K
    Kumari Monika1
    S
    S. Indu Kumari1,*
    1Division of Applied Biology, CSIR-Indian Institute of Chemical Technology, Tarnaka, Hyderabad-500 007, Telangana, India.
    2Academy of Scientific and Innovative Research (AcSIR), Ghaziabad-201 002, Uttar Pradesh, India.

    ABSTRACT

    Background: Sodium azide is a threat to the ecosystem and agriculture due to its high toxicity and increasing production. It is known to reduce seed germination in many plants with unknown mechanism of action.

    Methods: Seeds of Vigna radiata were treated with different concentrations of sodium azide using 0.1 M phosphate buffer pH 3.2 as a vehicle and control. Using seed germination assay data, 3 mM sodium azide was selected for further study which includes estimation of proteins, reducing sugar, α-amylase gene expression and activity, antioxidant activity and mineral analysis.

    Result: Our findings show that sodium azide at 2 mM concentration and above reduced Vigna radiata seed germination significantly. The treatment of Vigna radiata seeds with 3 mM sodium azide for 3 h showed physiological changes in germination affecting protein and reducing sugar content. These changes can be related to the low α-amylase activity in seeds treated with 3 mM sodium azide. Compared to control, treated seeds showed significant reduction in α-amylase activity per seed by 29, 40 and 41% at 30oC incubation for 24, 48 and 72 h respectively. This reduction in enzyme activity is supported by the inhibition of α-amylase gene expression. 3 mM sodium azide treatment causes reduction in antioxidant defense of germinating seeds by reducing the total phenolic content and DPPH quenching activity compared to control and vechile control treated seeds. Hence, it can be concluded that α-amylase inhibition at 3 mM sodium azide, along with oxidative stress, leads to germination failure.

    INTRODUCTION

    Seed germination, essential for plant development, involves changes in transcript, protein and hormone levels (Bewley et al., 1997). Plant hormones (gibberellic acid, abscisic acid) and enzymes like α-amylase regulate this process (Urbanova et al., 2016). α-amylase breaks down starch into sugars for energy (Kaneko et al., 2002). In Jatropha curcas, carbohydrate levels decrease during germination, indicating high utilization (Lopes et al., 2013). Free radical production is a stress factor, emphasizing the role of antioxidants and enzymes in successful germination (Bailly, 2004).
           
    Various organic and inorganic substances released into the environment disrupt plant life cycles and harm the ecosystem. Sodium azide (NaN3), a highly toxic compound with an unclear mode of action, has seen increased use over the past 30 years in airbags, pest control, fumigation (Tat et al., 2021) and as a mutagen (Jahan et al., 2020). At the cellular level, SA affects cell functioning by inhibiting mitochondrial cytochrome C oxidase and catalase (Keilin and Hartree, 1934; Stannard and horecker, 1948). Several studies on SA show that it inhibits seed germination in tomato (Adamu and Aliya, 2007), Eruca sativa (Khan et al., 2009), mungbean (Ali et al., 2024) and rice (Awan et al., 1980) which badly affect their growth and survival. It’s mode of action preventing seed germination is unknown. In this study the influence of SA upon the levels of macromolecules, gene expression and the activity of α-amylase enzyme, antioxidant defence and changes in mineral content during seed germination were recorded. These findings suggest that SA causes oxidative stress and reduces α-amylase activity, which resulted in failure of seed germination.

    MATERIALS AND METHODS

    Plant material
     
    The Vigna radiata seeds (IPM-2-14) were procured from Indian Institute of Pulse Research Kanpur, India. 
           
    The entire experiment was conducted in 2022-2024 at the Applied Biology Division, CSIR- Indian Institute of Chemical Technology, Hyderabad, Telangana.
     
    Germination treatment and Seed germination assay
     
    Vigna radiata (IPM-2-14) seeds were rinsed with autoclaved water, surface sterilized with 70% ethanol for 2 min and rinsed thrice to remove ethanol. Seeds were soaked in autoclaved water for 14 h at 25oC to initiate germination, then treated for 3 h at room temperature on a shaker with SA (1-10 mM in 0.1 M phosphate buffer, pH 3.2), water (control), or buffer (vehicle control). Treated seeds were washed, placed on moist filter paper in sterile petriplates and incubated at 30oC. Germination was recorded daily for 6 days. Per cent germination was calculated every 48 h and the SA concentration causing ~50% germination on day 4 was used for toxicity analysis.
     
    Estimation of reducing sugars and proteins
     
    Crude extract preparation was as per Sottirattanapan et al. (2017). Crude extract reducing sugars and protein content were estimated by DNS (3, 5-dinitrosalicylic acid) and Bradford methods respectively.
     
    Qualitative assay for α-amylase
     
    The Vigna radiata seeds used in the study were treated with SA, VC and control. Embryo less half seeds were tested to analyze the qualitative reduction in α-amylase activity on treating with SA (Liu et al., 2018). The α-amylase activity which hydrolyzed the starch is represented by the colorless zone.
     
    Quantitative assay for α-amylase
     
    The treated, control and VC Vigna radiata seeds were collected after incubation at 30oC for 24, 48 and 72 h. Five seeds from each treatment were homogenized using glass mortar and pestle in pre-chilled 10 ml of 0.1 M sodium acetate buffer pH 5.6, kept at 4oC for 10 min then centrifuged at 10,000 rpm for 10 min at 4oC. The supernatant was used for quantification of α-amylase activity. Quantification of α-amylase was done by DNS method of Miller (1959). Maltose was used as a standard to estimate the reducing sugars released due to starch degradation by α-amylase enzyme in crude extract using a spectrophotometer at 540 nm. One unit of α-amylase activity was defined as the quantity of enzyme required to release 1 µM of maltose per minute.
     
    Gene expression of α-amylase
     
    RNA isolation
     
    Seeds treated with 3 mM SA, VC and control were transferred to autoclaved petriplates layered with moist filter paper and kept at 30oC in oven followed by RNA isolation at intervals of 0, 24, 48 and 72 h after treatment by method of Meng and Feldman (2010).  The RNA pellet was dissolved in 50 μl DEPC-treated, autoclaved ddH2O and stored at -80oC.
     
    cDNA synthesis and qR-PCR
     
    Total extracted RNA was quantified with Nanodrop (Thermo Scientific-NanoDrop™ 2000c Spectrophotometer). Further 1 µg of total RNA was used to prepare cDNA using TaKaRa Prime Script™ 1st strand cDNA Synthesis Kit (Cat. #6110A) according to the manufacturers protocol. Quantitative Real Time PCR (q-RT PCR) was performed using TaKaRa TB Green® Premix Ex Taq™ II (Tli RNaseH Plus) (Cat. #RR820A) according to kit specified standard protocol. Comparative CT Value method (2-ΔΔCt) was used for the quantification of Gene expressions (Balaji et al., 2016).

    Free radical scavenging assay and total phenolic content
     
    Extract was prepared according to method of Wei et al. (2019) to estimate free radical scavenging activity and total phenolic content. To estimate free radical scavenging activity 2 ml of 0.2 mM DPPH in ethanol was mixed with 0.5 ml of extract. This mixture was incubated in dark for 30 min at room temperature. Absorbance was measured at 570 nm using spectrophotometer. Total phenolic content was estimated according to method of Xu and Chang (2007). The total phenolic content was expressed as gallic acid equivalents (mg of GAE/g sample) through the calibration curve of gallic acid.
     
    Mineral analysis
     
    Seeds of green gram were treated as mentioned above with 3 mM SA, Control and VC. Seeds were thoroughly washed with autoclaved distilled water and incubated at 30oC for 24 and 48 h. After incubation samples were prepared according to Alkaltham et al. (2020) to analyse calcium (Ca), potassium (K), sodium (Na), iron (Fe),  copper (Cu), zinc (Zn), magnesium (Mg) and manganese (Mn) using ICP-OES (CSIR-IICT, Hyderabad, India).
     
    Statistical analysis
     
    Two-way ANOVA was performed for obtaining statistical significance. Graph-pad prism software version 8.0.2 was used for all statistical tests and plotting the graphs of gene expression studies. One-way analysis of variance (ANOVA) followed by Tukey test was performed by using instat 3 software for analysis of other data. All tests were performed in at least three replicates and the data are expressed as means ± standard deviation (SD). Significant difference compared to control for same time period are indicated by *(P<0.05), **(P<0.01) and ***(P<0.001).

    RESULTS AND DISCUSSION

    Seed germination assay
     
    On 4 day, per cent seed germination data obtained was same as control and VC for 1 mM of SA. Per cent germination was reduced up to 82%, 38%, 23%, 00% for 3 h treatment of 2, 3, 4 and 5 mM SA respectively (Fig 1). These results revealed that, SA at concentration of 2 mM and above significantly affected the germination of Vigna radiata seeds. SA at the concentration of 5 mM and above showed complete reduction in germination of Vigna radiata seeds.

    Fig 1: Effect of sodium azide on green gram seed germination.


     
    Biomolecular assay
     
    Reducing sugars in SA, VC and control-treated seeds were quantified using the 3, 5-Dinitrosalicylic acid method Miller (1959). The control and VC showed a significant increase in reducing sugars At 24, 48 and 72 h , while SA-treated seeds did not (Fig 2A). Protein content, analyzed by the Bradford method showed no significant difference immediately after treatment. At 24 and 48 h, control and VC had reduced protein, while SA-treated seeds showed no change (Fig 2B).

    Fig 2A: Effect of sodium azide treatment on reducing sugar content of germinating seeds.



    Fig 2B: Effect of sodium azide treatment on protein content of germinating seeds.



    α-amylase gene expression study
     
    Gene expression analysis at 0 h condition of 3 mM SA in acidic phosphate buffer (pH-3.2) treated Vigna radiata seeds showed similar expression of α-amylase as in control and VC treated seeds. The expression of α-amylase down regulated in 3 mM treated seeds compared to the control and VC under 24 h condition. However, an up regulated expression of α-amylase was observed in VC, compared to the control samples at 48 h. The expression of α-amylase was found to decline in seeds treated with 3 mM SA with the increasing incubation time (Fig 3).

    Fig 3: Differential expression of α-amylase.


     
    α-amylase activity
     
    The Vigna radiata seeds treated with 3 mM SA for 3 h, incubated for 24, 48 and 72 h to evaluate effect of SA on the α-amylase activity.
     
    Quantitative assay
     
    Quantitative assay for α-amylase reveals that 3 mM SA treatment significantly decreased α-amylase activity as compared to control and VC. Immediately after treatment no significant differences were observed between α-amylase activity of control, VC and treated seeds. As compared to the control, seeds treated with 3 mM SA showed reduction in α-amylase activity by 29%, 40% and 41% at 24, 48 and 72 h incubation period at 30oC respectively. As compared to the VC, seeds treated with 3 mM SA showed reduction in α-amylase activity by 23%, 52% and 34% at 24, 48 and 72 h incubation period at 30oC respectively (Fig 4A).

    Fig 4A: Effect of Control, vehicle control and sodium azide on á-amylase activity in germinating seeds of green gram.


     
    Qualitative assay
     
    Qualitative assay was performed to assess the effect of SA on the enzyme activity after incubation of 48 h. The control and VC seeds showed greater more transparent area around them as compared to seeds treated with SA (Fig 4B).

    Fig 4B: Effect of Control, vehicle control and sodium azide on á-amylase activity in germinating seeds of green gram.


     
    Free radical scavenging activity and total phenolic content
     
    % DPPH scavenging activity in 3 mM treated seeds was significantly less as compared to control and VC treated seeds after 24, 48 and 72 h of incubation after post treatment (Table 1). Total phenolic content of control and VC treated seeds increased after 72 h of incubation but comparatively less increase was observed in 3 mM treated seeds (Table 1).

    Table 1: % DPPH scavenging activity and total phenolic content (TPC) of 3 mM SA ( sodium azide), control and VC ( vehicle control) treated seeds at different time incubation.


     
    Mineral analysis
     
    Seeds treated with 3 mM SA, control and VC as mentioned above were tested for mineral changes in such as Ca, Na, K, Fe, Cu, Zn, Mg and Mn during seed germination. Out of these eight tested minerals only Ca, Na and Fe showed statistically significant differences for the mentioned treatments. SA treatment showed rise in Ca content of treated seeds at 0 h of treatment compared to VC and control treated seeds while after 48 h of SA treatment Ca content in both VC and SA treated seeds decreased compared to control which was statistically significant (Table 2). An increase in Fe content after 24 h of treatment in SA treated seeds compared to control and VC treated seeds and with normal levels at 48 h of incubation was noted (Table 2). Na content in SA treated seeds was higher compared to both control and VC treated seeds at 0 and 48 h of incubation with a statistically significant rise as compared to control (Table 2).

    Table 2: Mineral content (mg/100 g) of 3 mM SA (sodium azide), control and VC ( vehicle control) treated seeds at different time incubation.


           
    Seed germination assay
     
    Seed germination inhibition by SA at concentrations ≥ 1 mM had been reported in Lycopersicon esculentum (Adamu and Aliya, 2007) and Eruca sativa (Khan et al., 2009). Our study on Vigna radiata showed a concentration-dependent decline in germination with increasing SA levels. Germination dropped significantly at 2 mM and was completely inhibited (0%) at 5 mM. These results align with the previous findings in Cajanus cajan (Chaudhary et al., 2021), confirming SA’s strong inhibitory effect.
     
    Biomolecular changes
     
    Seeds treated with 3 mM SA showed declined reducing sugar content, indicating impaired starch hydrolysis. Starch breaks down into glucose and maltose, providing energy for germination (Kaneko et al., 2002). Normally, germination involves starch breakdown for energy and protein degradation into amino acids (Palmiano and Juliano, 1972). In control and VC seeds, protein content decreased over time, while SA-treated seeds showed no significant change, suggesting inhibited protein reserve use. Similar trends were noted in grassland species, with rising sugar and declining protein levels during germination (Zhao et al., 2018). These effects may result from reduced α-amylase activity, crucial for starch breakdown and germination.
     
    α-amylase activity
     
    SA-treated seeds showed reduced gene expression and α-amylase activity, while VC seeds showed increased expression at 48 h. Combined SA and VC treatment confirmed SA’s inhibitory effect. α-amylase is vital for breaking down reserves during germination (Damaris et al., 2019), matching our observation as small transparent zones around the treated seeds in our study. Control and VC seeds showed rising enzyme activity unlike in SA-treated seeds. Similar reductions were observed under NaCl stress in rice (Liu et al., 2018) and during germination in rice and peas (Murata et al., 1968; Juliano and Varner, 1969).  Quantitative data supported qualitative results. In Pisum sativum, cadmium reduced germination via low amylase activity (Chugh and Sawhveey, 1996). Likewise, our study with SA treatment had led to high protein, low sugar, limited energy with failed germination. These findings match Ashraf et al. (2002), who linked reduced α-amylase activity to poor germination under salinity.
     
    Antioxidant activity
     
    The imbalance between excessive generation of reactive species and antioxidant defence leads to oxidative stress which disturbs the redox homeostasis, damages membrane lipids, proteins and nucleic acids, killing the plant cell (Jain and Shakkarpude, 2024). ROS interacts with various enzymes and inhibits them in turn affecting the physiology of the plant. Our study shows parallel reduction in both antioxidant defence and α-amylase activity due to SA stress which is similar to Zang et al. (2008). Our results show that SA treatment affected seed germination in Vigna radiata by reducing free radical scavenging activity and total phenolic content which act as protective measures of the cell during oxidative stress.
     
    Mineral analysis
     
    SA treatment increased Ca2+ levels by 44%, suggesting an oxidative stress. Pang and Wang (2008) noted that ionic stress leads to ROS accumulation, countered by antioxidants. An antioxidant failure damages the plant growth. Elevated Na in SA-treated seeds at 0, 24 and 48 h indicates failed germination. Salt stress can increase ABA, reduce gibberellic acid, hinder water uptake and cause toxicity, all of which impair seed germination and development (Nikolić et al., 2023). Excessive iron triggers oxidative stress and reduced growth in Chlorella vulgaris (Estevez et al., 2001). Our data shows increased Fe in SA-treated seeds after 24 h. Calcium, Sodium and Iron accumulation may cause ROS buildup, damaging seeds due to reduced oxidative defence.

    CONCLUSION

    Our study concludes that at 3 mM SA treatment seed germination in Vigna radiata and α-amylase enzyme activity was reduced, which in turn resulted in failure of starch and protein reserve utilization for their development. This reduction in enzyme activity is related to its low gene expression which suggests that SA might interfere at transcriptional level of α-amylase gene expression. Further due to SA treatment elevation in Ca, Fe and Na levels were recorded which could be responsible in causing oxidative damage. We conclude, hampered biochemistry of SA treated seeds could be a mixed response of elevation in Ca, Fe and Na, oxidative stress, reduction in antioxidant defence and inhibition of α-amylase enzyme.

    ACKNOWLEDGEMENT

    We thank the Director, CSIR-Indian Institute of Chemical Technology (IICT) (Ms. No. IICT/Pubs./2022/290.) For providing all the required facilities to carry out the project work. Masurkar Ajit Sopan (SRF) and Phadtare Aniket Pramod (JRF) are grateful to the University Grant Commission (UGC) for the award of the fellowship.

    CONFLICT OF INTEREST

    The authors declare that there are no conflicts of interest related to this research.

    REFERENCES


    1. Adamu, A.K. and Aliyu, H. (2007). Morphogical effects of sodium azide on tomato (Lycopersiconesculentum Mill). Science World Journal. 2(4): 9-12. doi: 10.4314/swj.v2i4.51755.

    2. Ali Basid, S.M.D., Konjengbam, S.N., Ahmad, F., Sanasam, S. and Kumawat, R. (2024). Ascertaining lethal dose 50 (LD50) and simultaneous effect of ethyl methane sulphonate (EMS) and sodium azide (SA) on seedling characters in mungbean genotypes 'Pusa 1031' and 'Pusa 1431'. Legume Research. 47(11): 1929-1935. doi: 10.18805/LR-5255.

    3. Alkaltham, M.S., Salamatullah, A.M., Özcan, M.M., Uslu, N. and Hayat, K. (2020). The effects of germination and heating on bioactive properties, phenolic compounds and mineral contents of green gram seeds. LWT. 134: 110106.

    4. Ashraf, M.Y., Sarwar, G., Ashraf, M., Afaf, R. and Sattar, A. (2002). Salinity induced changes in α-amylase activity during germination and early cotton seedling growth. Biologia Plantarum. 45(4): 589-591.

    5. Awan, M.A., Konzak, C.F., Rutger, J.N. and Nilan, R.A. (1980). Mutagenic effects of sodium azide in rice 1. Crop Science. 20(5): 663-668.

    6. Bailly, C. (2004). Active oxygen species and antioxidants in seed biology. Seed Science Research. 14(2): 93-107.

    7. Balaji, S.A., Udupa, N., Chamallamudi, M.R., Gupta, V. and Rangarajan, A. (2016). Role of the drug transporter ABCC3 in breast cancer chemoresistance. PloS one. 11(5): e0155013.

    8. Bewley, J.D. (1997). Seed germination and dormancy. The plant cell. 9(7): 1055.

    9. Chaudhary, L., Sharma, R. and Kumar, M. (2021). Estimation of LD50 and effect of sodium azide on germination and seedling parameters of different cultivars of Cajanus cajan (L.) Millspaugh. Toxicology and Environmental Health Sciences. 13(3): 279-285.

    10. Chugh, L.K. and Sawhney, S.K. (1996). Effect of cadmium on germination, amylases and rate of respiration of germinating pea seeds. Environmental Pollution. 92(1): 1-5.

    11. Damaris, R.N., Lin, Z., Yang, P. and He, D. (2019). The rice alpha-amylase, conserved regulator of seed maturation and germination. International Journal of Molecular Sciences. 20(2): 450.

    12. Estevez, M.S., Malanga, G. and Puntarulo, S. (2001). Iron-dependent oxidative stress in Chlorella vulgaris. Plant Science. 161(1): 9-17.

    13. Jahan, R., Malik, S., Ansari, B.S. and Khan, S. (2020). Evaluation of optimal doses for gamma rays and sodium azide in linseed genotypes. Agricultural Science Digest. 41(Special Issue): 207- 210. doi: 10.18805/ag.D-5165.

    14. Jain, A. and Shakkarpude, J. (2024). Oxidative stress: A biomarker for animal health and production: A review. Indian Journal of Animal Research. 58(1): 01-12. doi: 10.18805/IJAR.B-5300.

    15. Juliano, B.O. and Varner, J.E. (1969). Enzymic degradation of starch granules in the cotyledons of germinating peas. Plant Physiology. 44(6): 886-892.

    16. Kaneko, M., Itoh, H., Ueguchi-Tanaka, M., Ashikari, M. and Matsuoka, M. (2002). The α-amylase induction in endosperm during rice seed germination is caused by gibberellin synthesized in epithelium. Plant Physiology. 128(4): 1264-1270.

    17. Keilin, D. and Hartree, E.F. (1934). Inhibitors of catalase reaction. Nature. 134(3398): 933-933.

    18. Khan, S. and Al-Qurainy, F. (2009). Mutagenic effect of sodium azide on seed germination of Eruca sativa (L.). Australian Journal of Basic and Applied Sciences. 3(4): 3081-3087.

    19. Liu, L., Xia, W., Li, H., Zeng, H., Wei, B., Han, S. and Yin, C. (2018). Salinity inhibits rice seed germination by reducing α-amylase activity via decreased bioactive gibberellin content. Frontiers in Plant Science. 9: 275.

    20. Lopes, L.D.S., Gallão, M.I. and Bertini, C.H.C.D.M. (2013). Mobilisation of reserves during germination of Jatropha seeds. Revista Ciência Agronômica. 44: 371-378.

    21. Meng, L. and Feldman, L. (2010). A rapid TRIzol based two step method for DNA free RNA extraction from Arabidopsis siliques and dry seeds Weinheim: WILEY VCH Verlag. 5(2): 183-186.

    22. Miller, G.L. (1959). Use of dinitrosalicylic acid reagent for determination of reducing sugar. Analytical Chemistry. 31(3): 426-428.

    23. Murata, T., Akazawa, T. and Fukuchi, S. (1968). Enzymic mechanism of starch breakdown in germinating rice seeds I. An analytical study. Plant Physiology. 43(12): 1899-1905.

    24. Nikolić, N., Ghirardelli, A., Schiavon, M. and Masin, R. (2023). Effects of the salinity-temperature interaction on seed germination and early seedling development: a comparative study of crop and weed species. BMC Plant Biology. 23(1): 446.

    25. Palmiano, E.P. and Juliano, B.O. (1972). Biochemical changes in the rice grain during germination. Plant Physiology. 49(5): 751-756.

    26. Pang, C.H. and Wang, B.S. (2008). Oxidative stress and salt tolerance in plants. Progress in Botany. 231-245.

    27. Sottirattanapan, P., Nantachai, K., Daduang, S., Funahashi, T. and Yamada, M. (2017). Purification and characterization of amylase from roots of Paederiafoetida Linn. Biocatalysis and Agricultural Biotechnology. 10: 329-335.

    28. Stannard, J.N. and Horecker, B.L. (1948). The in vitro inhibition of cytochrome oxidase by azide and cyanide. J. Biol Chem. 172(2): 599-608.

    29. Tat, J., Heskett, K., Satomi, S., Pilz, R.B., Golomb, B.A. and Boss, G.R. (2021). Sodium azide poisoning: A narrative review. Clinical Toxicology. 59(8): 683-697.

    30. Urbanova, T. and Leubner Metzger, G. (2016). Gibberellins and seed germination. Annual Plant Reviews. 49: 253-284.

    31. Wei, Y., Wang, X., Shao, X., Xu, F. and Wang, H. (2019). Sucrose treatment of mung bean seeds results in increased vitamin C, total phenolics and antioxidant activity in mung bean sprouts. Food Science and Nutrition. 7(12): 4037-4044.

    32. Xu, B.J. and Chang, S.K.C. (2007). A comparative study on phenolic profiles and antioxidant activities of legumes as affected by extraction solvents. Journal of Food Science. 72(2): S159-S166.

    33. Zhang, H., Hu, L.Y., Hu, K.D., He, Y.D., Wang, S.H. and Luo, J.P. (2008). Hydrogen sulfide promotes wheat seed germination and alleviates oxidative damage against copper stress. Journal of Integrative Plant Biology. 50(12): 1518-1529.

    34. Zhao, M., Zhang, H., Yan, H., Qiu, L. and Baskin, C.C. (2018). Mobilization and role of starch, protein and fat reserves during seed germination of six wild grassland species. Frontiers in Plant Science. 9: 234.
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