Nitric Oxide and Hydrogen Peroxide Interaction

Abstract

The potential function of hydrogen peroxide (H2O2) and nitric oxide (NO) in the induction of cadmium (Cd) stress tolerance in cyanobacteria has been well documented in the current work. To examine several physiological and biochemical parameters in this regard, Nostoc muscorum and Anabaena sp. subjected to Cd (6 M) stress were treated with H2O2 and SNP (sodium nitroprusside, NO donor). The results showed that Cd treatment decreased growth, pigment content, photosynthetic oxygen yield, and PS II photochemistry performance (increased energy flux parameters, such as ABS/RC, TRo/RC, ETo/RC, DIo/RC, and Fo/Fv, and decreased chlorophyll a fluorescence parameters, such as Po, o, and eo). Similar to how glutamate dehydrogenase (GDH) activity showed a reverse trend, uptake of nitrate (NO3-) and nitrite (NO2-), as well as the activities of nitrate and ammonia absorbing enzymes as well as carbohydrate content, were all negatively impacted by Cd poisoning. A relatively low dose (1 M) of H2O2 (only for 3 hours) and NO (SNP; 10 M) applied exogenously significantly reduced the toxicity caused by the Cd.

Introduction

By directly converting atmospheric nitrogen into ammonia, heterocystous, photosynthetic cyanobacteria in rice fields perform as biofertilizers and sustain the paddy crops. The most lucrative and common species that live in paddy fields are Nostoc muscorum and Anabaena sp. (Singh et al. 2016; Tiwari and Prasad 2020). They are regarded as one of the essential elements for sustainable agricultural development since they can fix atmospheric nitrogen on average at 20 to 25 kg per ha each season. In addition, they can increase the soil's capacity for aeration and produce a variety of advantageous extracellular products as well as certain hormones that promote plant growth (Prasanna et al. 2013; Chittora et al. 2020). Additionally, they can help our main crops withstand a variety of biotic and abiotic challenges (Saadatnia and Riahi 2009; Chittora et al. 2020). As a result, the lush growth of cyanobacteria in rice fields is crucial for the sustainability of agriculture, and an increase in rice yield may help to meet the food needs of our rapidly expanding population.

Materials and Methods

Organisms Used in Tests and Cultivation Conditions

The homogenous, filamentous, and heterocystous cyanobacteria Nostoc muscorum ATCC 27893 and Anabaena sp. PCC 7120 were cultured in BG-11 medium (pH 7.5) in a temperature-controlled room having 25 ± 2 °C under 75 μmol photons m−2 s−1photosynthetically active radiation (PAR, 400-700 nm) provided by white fluorescent tubes (Osram L 40 W/25-1) with a 14:10 h regime of light: dark. The cultures of the exponential phase were used to conduct the studies.

Designing Experiments and Treating Cultures

Both of the examined cyanobacteria's exponential phase cultures were collected for all experiments by centrifuging them at 3,000 g for 15 min. The cells were then twice washed with sterile distilled water. Henceforth, cyanobacterial cells were suspended in a growth medium containing Cd (6 µM), 10 µM SNP (sodium nitroprusside; a donor of NO), 20 µM PTIO (2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide; scavenger of NO), 100 µM LNAME (Nω-Nitro-L-arginine methyl ester hydrochloride; NOS enzyme's inhibitor), 1 µM H2O2, 1 mM NAC (N-acetyl-L-cysteine; scavenger of H2O2), 1 µM DPI (diphenyleneiodonium chloride; an inhibitor of NADPH oxidase enzyme) wherever required.

Evaluation of Growth

In order to analyse the optical density (OD) of the growth of the two test organisms, 3 ml of treated and untreated cells from each culture were harvested, and they were then thoroughly homogenised. The optical density of each set's culture was then measured at 750 nm using a Shimadzu, Japan, double beam UV-visible spectrophotometer.

Statistical Analysis

With the aid of SPSS 16.0, the statistical analysis of the outcomes was conducted using analysis of variance (ANOVA). When there were significant differences between the treatments at P 0.05 significance levels, the Tukey test was employed to separate the means. Results are presented as means with standard errors from three replicates (n = 3).

Conclusion

In conclusion, our findings indicated that the elimination of Cd stress from the cells of the cyanobacteria N. muscorum ATCC 27893 and Anabaena sp. PCC 7120 is significantly aided by the signalling molecules H2O2 and NO. Directly or indirectly, high Cd can have negative effects on important cellular metabolisms such as PS II photochemistry and nitrogen metabolic by causing excessive ROS production in cells. This stunts the growth of the test cyanobacteria. Additionally, even under Cd stress, exogenous administration of H2O2 and NO favourably regulated cellular metabolism and eliminated the detrimental effects. Therefore, it appeared that H2O2 and NO were the main sources of cyanobacteria N. muscorum and Anabaena sp.'s resistance to the toxicity of heavy metals, particularly Cd. The interdependence that these remarkable signalling molecules show for one another further indicates that the signalling pathway for these molecules is not linear. The current study makes it clear that H2O2 regulates NO, and that NO in turn leads to the regulation of several physiological and biochemical processes as well as genetic alterations inside the stressed organisms, making them better able to withstand environmental stress circumstances. The current study suggests using SNP, a relatively inexpensive source of NO, as a growth regulator in rice fields to encourage the luxuriant growth of cyanobacteria, an effective biofertilizer. By giving these cyanobacteria full resistance to Cd stress through exogenous supplementation of a very low dose of NO (SNP), the quality and yield of rice crops can be improved.

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