Our results showed that biofortification of lentil seedlings with ZnO NPs significantly increased the Zn concentration in the entire seedling, surpassing a 10-fold increase compared to the control condition. This finding is consistent with our recent report on the potential of ZnO NPs for Zn biofortification in mung bean seedlings8. For most crops, the recommended Zn concentration for proper growth is typically reported to be between 15 and 20 ppm of dry weight (DW) approximately13. According to Marschner14, most plant species naturally contain Zn concentrations between 30 and 100 ppm, while concentrations above 300 ppm DW are considered toxic. Our research findings align with these above-mentioned levels, highlighting the differing tolerance levels of plant organs towards toxicity. Specifically, we observed apparent signs of toxicity, such as reduced root and shoot biomass compared to the control, at Zn internal concentrations exceeding 300 ppm in the roots and 27.20 ppm in the shoot.
On the other hand, our data confirms the significant limitation of root-to-shoot Zn translocation which show the lentils root is more appropriate target for Zn biofortification. The ratio of root Zn concentration to shoot increased from 8.07 in the samples treated with 10 ppm to 24.52 in samples treated with 160 ppm ZnO NPs. The authors argue that this phenomenon is part of the plant’s strategy to sequester Zn into root vacuoles, preventing its accumulation in the highly metabolically active regions15,16,17. Despite the impressive ability of plants to sequester Zn in the roots, resulting in an imbalanced increase in Zn content in the shoots, the toxic effect of Zn on plant biomass persisted.
Similar to our previous findings in mung bean8; the current results also demonstrate a dose-dependent biphasic trend in the growth of lentil seedlings exposed to ZnO NPs. Specifically, at concentrations of 10 and 20 ppm, ZnO NPs had a beneficial impact on biomass (Table 1). Therefore, the positive effects of Zn on biomass, chlorophyll content, carbohydrates and proteins are probably due to its proven role in various biosynthetic processes from genetic materials to chlorophyll, carbohydrates and auxins and its role in enzyme activity18. Consequently, increased Zn content can impart advantageous effects on the plant, leading to improved growth. The positive effect of Zn application on chlorophyll content and photosynthesis has been reported in other studies conducted in other plants such as tomato19, mung bean8, Flax20 and Cilantro21. Additionally, the vital role of Zn in the formation of Pchlide and its contribution in chloroplast development was determined22.
On the other hand, a toxic effect was observed when the concentration of ZnO NPs exceeded 20 ppm. This was evident as the dry weight of lentil seedlings treated with ZnO NPs decreased, reaching its lowest level of 45.78% compared to the control at 160 ppm (Table 1). Additionally, the higher concentrations of ZnO NPs (40–160 ppm) likewise had a negative effect on the content of chlorophyll a and b. Researchers have demonstrated that excessive Zn in plant cells can cause toxicity by triggering various mechanisms. These include the generation of reactive oxygen species (ROS) which disrupts cell membrane integration and permeability, leading to the activation of oxidative stress signaling pathways23,24. Metal NPs, such as Zn NPs, have been found to deposit on the cell surface or within subcellular organelles, resulting in cellular oxidative stress25. According to reports, Zn toxicity disrupts the absorption and translocation of essential elements such as Fe and Mg into the chloroplast, affecting the functionality and efficiency of the photosynthetic system in various plant species. This results in the repression of PSII activity26.
MDA and H2O2 content can be considered as bioindicators for the oxidative stress. In current study, the ZnO NPs dependent increase of H2O2 and MDA content in the seedling confirmed oxidative damage (Fig. 3). Plants prevent the accumulation of ROS and subsequent lipid peroxidation by using enzymatic and non-enzymatic antioxidant systems.
The detoxification of ROS in plants involves several enzymatic reactions. Superoxide dismutase (SOD) plays a crucial role by converting superoxide anions into hydrogen peroxide (H2O2), while peroxidase (POX) further reduces hydrogen peroxide to water (H2O). PPO contributes to ROS detoxification by consuming available oxygen while catalyzing the oxidation of monophenols and/or o-diphenols, which results in the formation of o-quinones. As a byproduct of this reaction, PPO generates superoxide anions, which can be dismutated enzymatically by SOD. This dismutation eventually generates H2O2 as a secondary product, which serves as a substrate for the subsequent enzymatic activity of POX. Collectively, the sequential actions of PPO, SOD, and POX ensure the efficient detoxification of ROS in plants.
SOD plays an important role in counteract damaging effects of ROS by converting superoxide anion to a less toxic form of hydrogen peroxide. Zn is present in the cystolic and chloroplastic Cu/Zn-SOD enzymes, which play a critical role against the oxidative stress. Our findings are consistent with the results of other researchers27,28,29,30, who studied the effect of ZnO NPs on antioxidative enzymes in Moringa peregrine, Zea mays, Vigna mungo and Lectuca sativa, respectively.
The results related to the increase in the activity of the antioxidative enzymes such as SOD, POX and PPO in plants treated with ZnO NPs are in agreement with the findings obtained in our previous study, which showed that the treatment with ZnO NPs led to the increase in the activity of PPO and POX enzymes in mungbean plants8.
The differential response of PPO (Fig. 4D), with the highest activity at 40 ppm ZnO NPs, compared to POX (Fig. 4B) and SOD (Fig. 4C), which showed maximum activity at 160 ppm ZnO NPs, in the shoots of treated seedlings may be attributed to their distinct roles in plant defense mechanisms. SOD and POX primarily mitigate oxidative stress by scavenging ROS, whereas PPO is involved in the polymerization of phenolic compounds, which contributes to the strengthening of the cell wall and other defensive structures. The coordinated activity of POX and PPO in the roots of the treated plants may be related to their role in limiting the entry of excessive NPs into the plant by reinforcing the cell wall, a hypothesis that warrants further investigation. The content of H2O2, a product of SOD activity and a substrate for POX, correlates with the activity levels of these enzymes, with high H2O2 levels at 160 ppm signifying intense oxidative stress and triggering maximum SOD and POX activities to mitigate damage. An increase in phenolic, flavonoid and flavonol compounds was observed in the present study under the treatment of ZnO nanoparticles. The role of Zn in carbon allocating to the production of phenolic compounds in the shikimic acid and acetate cycle can be one of the reasons for the increase of these compounds31. The effect of NPs on phenolic compounds as ROS scavengers is related to their important role in the yield and plant adaptation to biotic and abiotic stresses32. The results of this study are consistent with the results of31,33,34 in the potato, Capsicum annuum and rapeseed plant, respectively.
Phenolic and flavonoid compounds are secondary metabolites that play a protective and antioxidant role in plants. Several studies showed that in plants treated with high concentrations of heavy metals, these compounds are active and protect the plants. Flavonols contained in the vacuoles can be used in the flavonol-peroxide cleansing system to scavenge reactive oxygen species, especially H2O2.
Similar to our findings, other studies have reported positive effects of low concentrations of ZnO NPs on protein content and plant growth in4 Solanum lycopersicum35,36 and Vignea radiata8. Intracellular proteins can be oxidized when exposed to free radicals and subsequently degraded by proteolytic enzymes. In the present study, the protein content in plants treated with lower concentrations (10 and 20 ppm) of ZnO NPs increased compared to the control. However, a decrease in protein content was observed in plants treated with higher (80 and 160 ppm) concentrations of ZnO NPs. This suggests that while Zn is essential for maintaining the balance of reactive oxygen species (ROS) and protein stability37, excess Zn can disrupt this balance, leading to reduced protein content, as seen in the present study.
Considering that the recommended daily intake of Zn in the diet is between 8 and 12 mg, with the consumption of about 10 g of 7 days old lentils seedlings, germinated and grown on culture containing 160 ppm of ZnO NPs, this need can be met. Thus, according the tremendous effect of ZnO NPs treatment on the Zn content of seedlings (about 14 times more than that of control), this method could lead to an augmentation that could meet the daily needs of adults.
The administration of ZnO NPs-biofortified lentil seedlings to rats demonstrated an increase in Zn content in blood serum, especially in the T2 group. This supports the bioavailability of Zn from ZnO NPs-treated plants, aligning with the findings of our previous report8. Furthermore, the observed increase in serum magnesium in both T1 and T2 groups suggests a potential positive influence of Zn-biofortified lentil seedlings on magnesium absorption or metabolism.
The decrease in serum iron levels in the control (+) group and a trend of lower iron levels in the T1 and T2 groups hint at a potential negative impact of lentil seedlings on iron absorption. Further investigations are needed to elucidate the underlying mechanisms.
Analysis of additional biochemical parameters, including triglycerides, cholesterol, glucose, and creatinine, revealed notable differences between experimental groups. In the T1 group, there was a significant increase in urea levels and a slight reduction in triglyceride levels. In the T2 group, there was a significant reduction in triglyceride levels and a significant increase in creatinine levels. The significant increase in creatinine levels in rats that received Zn-biofortified lentil seedlings raises questions about potential renal effects, warranting further exploration. These findings suggest that ZnO NP biofortification may have potential benefits for managing lipid profiles, but also highlight the need for further investigation into its impact on kidney function.Among the assayed enzymes (ALT, AST, and ALP), the T2 group showed a significant increase in AST activity compared to the control groups. The elevated AST activity in the T2 group indicates a potential impact on liver function. However, the lack of significant changes in ALT and ALP suggests a nuanced response that requires deeper investigation.
.png?w=768&resize=768,0&ssl=1 "How to Use Lentils to Add Fiber and Protein to Your Favorite Meals and Snacks")
