Effects of zinc oxide nanoparticles on growth and antioxidant enzymes of Capsicum chinense -
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Effects of zinc oxide nanoparticles on growth and antioxidant enzymes of Capsicum chinense
Introduction: The research paper investigates the impact of zinc oxide nanoparticles on the germination of Capsicum chinense seeds and the growth of resulting plants. The study found that suspensions of zinc oxide nanoparticles at concentrations between 100 and 500 mg L^-1 led to increased germination and improved development of plumule and radicle lengths. Additionally, the activities of peroxidase, catalase, and ascorbate peroxidase enzymes were measured, revealing increased activities of the former two enzymes at all concentrations, while the activity of the latter was reduced at 500 mg L^-1. The research suggests that pretreating seeds with ZnO nanoparticles can enhance germination, seedling development, and the activities of antioxidant enzymes. Nanomaterials, particularly zinc oxide nanoparticles (ZnO NPs), have shown promise in modifying conventional agricultural practices as fertilizers and pesticides. ZnO NPs have been found to influence plant anatomy, biochemistry, and physiology, acting as antimycotic agents and enhancing seed germination rates and plant growth. However, the effects of other metallic nanoparticles such as copper oxide, ferric oxide, titanium oxide, and cerium oxide on plant growth and development vary, with both positive and negative outcomes reported. The positive effects of nanoparticles on plants are attributed to the alterations in their physical, chemical, and biological properties, which in turn impact chemical and biological activities in plants. Consequently, this study aimed to investigate the effects of ZnO NPs on seed germination, seedling growth, and antioxidant enzymes of Capsicum chinense (habanero pepper) due to its increasing agricultural significance and the lack of prior research on its responses to ZnO NPs.
Material and Methods: Characterization of nanoparticles The characterization of nanoparticles in the research paper involved ZnO nanoparticles obtained from Nanostructured and Amorphous Materials Inc. The characterization process included morphological and structural analysis using transmission electron microscopy (TEM) and high- resolution TEM (HRTEM). Additionally, selected area diffraction (SAED) was performed using an electron microscope at 300 kV. The specific microscope used for these analyses was the FEI- TITAN 80-300 kV from Fisher Scientific. Furthermore, the TEM and HRTEM micrographs were processed using fast Fourier transform software, specifically Digital Micrograph 3.7.0 from Gatan Software. This comprehensive characterization approach allowed for a detailed examination of the nanoparticles’ morphology and structure, providing valuable insights into their properties.
Seed germination and seedling growth: The study investigated the effect of zinc oxide nanoparticles (ZnO NPs) on seed germination and seedling growth. Different concentrations of ZnO NPs (ranging from 100 mg L^-1 to 500 mg L^-1) were applied to the seeds during the imbibition stage. The seeds were then placed in a test chamber for 72 hours at 25°C with a photoperiod of 16 hours light and 8 hours darkness. After the imbibition stage, four replicates of 25 seeds per treatment were planted on Anchor paper and placed in polyethylene terephthalate baskets in the test chamber for 14 days. The bioassay followed the International Seed Testing Association (ISTA) rules, and the vigor (V%) and germination rate (G%) were determined at 7 and 14 days after seeding. Additionally, the fresh biomass per seedling was measured at the end of the 14-day period. Normal seedlings were defined as those with a root and plumule, each with a development of 2.0 cm. The length of the plumule and radicle were measured, and the fresh biomass of normal seedlings was determined using an analytical balance.
Overall, the study focused on the impact of ZnO NPs on seed germination and seedling growth, and the parameters assessed included vigor, germination rate, and biomass. The findings from the bioassay provided insight into the potential effects of different concentrations of ZnO NPs on seedling development.
Enzymatic activities: In the Enzymatic activities section of the research paper, the authors describe the process of assessing enzymatic activities in plantlets. The plantlets were frozen with liquid nitrogen at the end of the assays and then processed to determine the activity of three key enzymes: peroxidase (POD), ascorbate peroxidase (APX), and catalase (CAT). To extract the proteins for analysis, 200 mg of tissue (plumule and radicle) were used. The extraction process involved grinding the tissue with liquid nitrogen and polyvinylpyrrolidone in a pre-frozen mortar. Subsequently, the samples were collected in centrifugation microtubes, to which 1 mL of extraction buffer (phosphate buffer 100 mmol L À1 , pH 7, supplemented with EDTA 0.1 mmol L À1 ) was added. The mixtures were then centrifuged at 1200 rpm for 5 min at 4°C, and the resulting supernatants were stored at -20°C for further analysis. The authors detailed the specific techniques and conditions used for assessing the enzymatic activities, providing a clear and systematic methodology for their research. This section of the paper outlines the precise steps and conditions employed to determine the activity of POD, APX, and CAT enzymes in the plantlets, serving as an important contribution to the overall findings of the study.
POD activity: The research paper describes the determination of peroxidase (POD) activity using pyrogallol as a substrate. The reaction involved a 3 mL volume containing 15 µL of protein extract, 2.5 mL phosphate buffer (pH 8, 100 mmol L^-1), 320 µL of 5% pyrogallol, and 165 µL of H2O2 to 0.147 mmol L^-1. The reaction was initiated by adding H2O2, and the increase in absorbance was measured at 420 nm every 20 seconds for 1 minute. One unit of POD activity was defined as 1.0 mg of purpurogallin formed in 20 seconds at pH 6 with an absorbance coefficient of 26.6 mM^-1 cm^-1.
The method used in the experiment provides a standardized and quantifiable measure of the POD activity present in the protein extract. The specific conditions, including the pH level and time frame for measurement, are clearly outlined, ensuring reproducibility of the results. The use of pyrogallol as a substrate and H2O2 as the initiator aligns with established methods for measuring peroxidase activity. These findings contribute to the understanding of POD activity and can be applied in various research areas, particularly in the analysis of enzyme kinetics and the investigation of oxidative stress responses in biological systems.
Experimental design and statistical analysis :The experimental design used in this study involved a completely randomized design with six treatments and four repetitions. The general linear model utilized in the analysis included the response variable Yij, the overall average, the effect of ZnO NPs concentrations, and the experimental error. The experimental unit consisted of an Anchor paper roll containing 25 habanero seeds. Data assessments were conducted using analysis of variance and the average comparison of treatments by the Tukey test (p0.05) using SPSS Statistics, 21 Version. To evaluate the possible linear, quadratic, or cubic trends of each variable and treatment, an orthogonal polynomial analysis was performed. In this analysis, the trends L, Q, and C were considered significant at a level of p0.05.
Results: The results and discussion section of the research paper presents findings related to the characterization of zinc oxide nanoparticles (ZnO NPs). Transmission electron microscopy (TEM) images revealed the quasi-spherical morphology of the NPs, with lattice strips exhibiting a spacing of 2.81 Å corresponding to lattice planes {100} of the ZnO hexagonal structure. The size distribution analysis indicated that 75% of the particles had diameters within the range of 12-24 nm, with 30% of the NPs falling between 12 nm and 20 nm. Additionally, the selected area electron diffraction (SAED) pattern displayed irregular rings associated with planes (100), ( ), ( ), and (110) of the crystalline structure. These results provide valuable insights into the morphological and structural characteristics of the ZnO NPs, laying the groundwork for further discussions on their potential applications and associated properties.
Effect of ZnO NPs on seed germination and vigor:
The research investigated the effect of ZnO nanoparticles (NPs) on seed germination and vigor. The study found that increasing concentrations of ZnO NPs resulted in improved seed vigor, with the highest increase observed at a concentration of 500 mg L^-1, exceeding the control treatment by 126%. Additionally, significant and quadratic trends were observed for germination and fresh biomass, with the maximum biomass attained at 400 mg L^-1, exceeding the control by 114%. Seedling growth increased with concentrations of 100-300 mg L^-1, with the highest growth observed at 300 mg L^-1, exceeding the control treatment by 250%. However, slight decreases were found at 400 and 500 mg L^-1. The research also reported that the presence of ZnO NPs during germination leads to hormone biosynthesis, promoting seed reserves mobilization and enzyme activation. The improvement of physiological seed quality was attributed to metallic NPs inducing photosterilization reactions and promoting water and oxygen imbibition necessary for faster germination. Furthermore, the use of nanomaterials in agriculture was highlighted for their positive impact on seedling germination and plant growth. Overall, the study demonstrated the potential of ZnO NPs to positively influence seed germination and seedling growth, with the effects being dependent on the concentration of NPs applied.
Plumule and radicle length: The study investigated the impact of zinc oxide nanoparticles (ZnO NPs) on the growth of plumule and radicle in seedlings. The results indicated that ZnO NPs positively influenced plumule length, with an optimal effect observed at 300 mg L À1. However, at higher concentrations (400 and 500 mg L À1), plumule lengths were slightly shorter. Similarly, radicle growth increased up to 300 mg L À1 but decreased at higher concentrations. The findings contrast with previous studies that demonstrated positive effects of ZnO NPs on seedling growth at lower concentrations. Importantly, the study suggests that the effect of ZnO NPs on plant physiology varies depending on the species and concentration, with lower concentrations inducing growth and higher concentrations inhibiting seedling growth. This highlights the complex and species-specific nature of the impact of ZnO NPs on plant development. The study contributes to our understanding of the dose-dependent effects of ZnO NPs on seedling growth and emphasizes the need for further research to elucidate the mechanisms underlying these contrasting effects.
Antioxidant enzyme activity: The study examined the effects of ZnO NPs on antioxidant enzyme activity in C. chinense seedlings. Results showed a significant increase in peroxidase (POD) activity with ZnO NPs treatment, particularly at a concentration of 400 mg/L, indicating stimulation of the antioxidant defense system. Additionally, concentration-dependent increases were observed in ascorbate peroxidase (APX) and catalase (CAT) activities. At 400 mg/L, APX activity increased four-fold compared to the control, while CAT activity at 500 mg/L exceeded control levels by 544%. Interestingly, as the concentration of reactive oxygen species (ROS) increased, APX activity decreased while CAT activity increased, suggesting an adaptive response of the antioxidant enzyme system to counteract excessive ROS production. The findings suggest that ZnO NPs can influence the balance of antioxidant defense systems in plants to mitigate oxidative stress and maintain enzyme function. This research highlights the potential phytotoxic effects of ZnO NPs and provides insights into their impact on the antioxidant enzyme system in plant seedlings.
Conclusions: The study found that treatments with ZnO nanoparticles (NPs) had a positive impact on germination, seed vigor, and seedling growth, indicating their potential as nanofertilizers in agriculture. The research observed that ZnO NPs in the range of 0 to 300 mg L^-1 improved plumule and radicle development, but concentrations of 400 and 500 mg L^-1 resulted in a decrease in these tissues, indicating potential toxicity at higher levels. High concentrations of ZnO NPs were found to be potentially toxic to the growth of habanero chili plants. The study also noted an increase in the activity of antioxidant enzymes (POD, APX, CAT) associated with oxidative stress as the concentration of ZnO NPs increased. These findings contribute to understanding the effects of ZnO NPs on germination, seed vigor, and potential phytotoxic effects on seedlings. Overall, the research suggests that while ZnO NPs have the potential to enhance agriculture as a nanofertilizer, careful consideration of concentration levels is essential to avoid potential toxicity to plant growth.