The study of incredibly small structures, with sizes ranging from 0.1 to 100 nanometers, is known as nanotechnology. It is a significant technological advancement that might significantly affect our lives in the ensuing decades[1]. The field of nanotechnology is really multidisciplinary, including the manipulation and control of individual atoms and molecules to create novel materials, nanomachines, and nanodevices that find application in several domains of human endeavors. The process of treating material at the nanoscale is known as nanotechnology[2]. Almost all disciplines have found that the field of nanotechnology is one of the most popular for research and development [3].

Because of their chemical and physical characteristics, zinc oxide nanoparticles are among the most extensively utilized nanomaterials and have attracted a lot of interest from the scientific, medical, and agricultural communities[4]. They are also useful in a variety of medicinal and antimicrobial applications. By supplying sufficient amounts of zinc, zinc plays a significant role in plant growth and metabolism as well as hormone control, glucose metabolism, protein synthesis, and stress reduction[5].Zinc nanoparticles have been demonstrated to stimulate seed plants, guard against illnesses and unfavorable growing circumstances, and have a good effect on the growth and development of mung beans[6]. Manufactured zinc oxide nanoparticles have been shown to improve agronomic and physiological parameters such plant height, leaf area, and pigment content, as well as effectively prevent the Cercospora leaf spot disease in mung beans[7, 8]. One of the most important variables that negatively impacts a plant's physiological processes and eventually lowers agricultural production is drought stress. A variety of morphological, physiological, chemical, and biochemical changes are brought about by water stress in plants, with the main goal being to decrease water loss through transpiration while attempting to improve water use efficiency in the plant[9]. Plants under water stress develop less quickly because it increases the generation of free radicals that oxidize cells. This process, known as oxidative stress, worsens the effects of water stress on growth markers [10].Additionally, it impacts photosynthesis, the relative water content (RWC) of the leaves, and the opening and shutting of stomata[10,11].ZnO-NPs were reported to benefit plants in water stress circumstances [12]. Zinc oxide nanoparticle treatment of water-stressed potato plants resulted in an increase in the pigments used in photosynthesis and gas exchange in the leaves. In addition to boosting the activity of antioxidant enzymes in peanut plants under water stress, foliar spraying with zinc oxide nanoparticles also enhanced biomass and the number of pods[13]. Increases in shoot length, root length, leaf count, and branch count were seen in mung bean plants treated with nanoparticles[14].

Mung bean (Vigna radiata L.) is a summer annual plant in the legume family Leguminosae, sometimes known as the green gram[15]. It has a short life cycle (75-90 days) and is drought resistant[16]. Legume crops, particularly mung beans, rank second behind cereal crops owing to their high nutritional content, which includes proteins, carbs, lipids, vitamins, and minerals. Mung bean seeds provide 24-28% protein, 59-65% carbs, 1-1.5% fat, and 3.5-4.5% fiber [17]. Mung beans may fix atmospheric nitrogen by creating symbiotic interactions with Rhizobium bacteria, which leads to higher soil fertility [18]. Mung beans are therefore a component of agricultural rotations, especially when they are planted alongside cereal crops like rice[19]. With a cultivated area of 13.84 thousand hectares, mung bean output in Iraq is still modest when compared to other nations, despite its importance [20]. The current study is to evaluate the impact of (ZnO NPs) on some physiological and biochemical properties of mung bean plants under water stress circumstances, given the significance of this crop.

On May 28, 2023, the current investigation was done in the Forestry Department's wooden shade structure at the College of Agriculture and Forestry, University of Mosul. The experiment entailed sowing 10 seeds at a depth of 1 cm from two mung bean kinds (Iraqi and Mexican) in plastic pots with a capacity of 5 kg. The soil was a loamy sand mix sourced from an agricultural region near Mosul (Sada and Ba'u'baiza). After 15 days of sowing, the number of seedlings was reduced to five per pot. After 40 days of planting, the plants were exposed to three levels of field capacity (80%, 50%, and 30%).Fifteen days after exposing the plants to water stress, They sprayed the plants with three different concentrations of ZnO nanoparticles (200, 100, and 0 parts per million) until thoroughly wet.

Irrigation was carried out using regular water, maintaining the field capacity levels of the soil by weighing the pots daily with a scale. After 60 days from the planting date, some physiological and biochemical indicators were studied:

1. Plant Pigment Content: Chlorophyll a and Chlorophyll b

2. Estimation of Antioxidant Enzyme Activity: Peroxidase (POD), Catalase (CAT), and Proline

3. Estimation of Carbohydrate Content

1- Plant Pigment Content: Chlorophyll a and Chlorophyll b   

1. Estimation of Chlorophyll a Content

The results shown in Table 1 indicate that treating mung bean plants with zinc nanoparticles at concentrations of 100 and 200 parts per million led to a notable rise in the amount of chlorophylla.At a concentration of 100 parts per million, this rise was most noticeable, reaching 41.25% in comparison to the control treatment. On the other hand, compared to the control plants, water stress significantly reduced the amount of chlorophyll a content, with a loss of 14.81% at 30% field capacity.. The Mexican variety of mung bean showed a clear superiority over the Iraqi variety. Regarding the interaction between varieties and concentration, the Mexican plants treated with 100 parts per million showed the best results compared to other treatments. The results of the three-way interaction (varieties × concentration × field capacity) revealed a major decrease in chlorophyll levels in the Iraqi variety that was not treated with nanoparticles and was grown under 30% field capacity condition

Table 1: ZnO Nanoparticles Used at Various Concentrations and Their Impact on the Chlorophyll a Content of Mung Beans Grown under Water Stress Conditions

2.1Estimation of Chlorophyll b Content:

Observing the results in Table 2, it is clear that spraying mung bean plants using nanoparticles of zinc oxide resulted in a significant increase in the amount of chlorophyll b at 100 and 200 ppm. The increases were 22.54% and 21.83%, respectively, compared to the control treatment. Regarding the effect of water stress, there was a noteworthy rise in chlorophyll b concentration with an uptrend in field capacity to 50% and 80%, with increases of 17.33% and 8.66%, respectively, compared to a field capacity of 30%.

Regarding the varieties, the results show that the Mexican variety significantly outperformed the Iraqi variety. The data on the two-way interaction (concentration × drought) indicate that spraying plants with nanoparticles, particularly at a concentration of 100 parts per million and grown at a moisture level of 80%, showed the best results. As for the three-way interaction (varieties × concentration × drought),at a moisture content of 80% and a ZnO-NPs concentration of 100 parts per million, the Mexican species yielded the greatest value

Table 2: Impact of using zinc oxide nanoparticles at different concentrations on the Chlorophyll bContent of Mung Bean grown under water stress conditions

2-Estimation of Antioxidant Enzyme Activity: Peroxidase (POD), Catalase (CAT), and Proline

2.1 Estimation of Proline 

From Table 3 it is evident that the application of various concentrations of ZnO NPs (0, 100, and 200) ppm caused a considerable drop in proline levels in the mung bean plants' leaf tissues. The treatment with 100 parts per million recorded the greatest decrease in this amino acid, showing a reduction of 30.42% in contrast to the control group treatment. In relation to the effects of water stress, the results indicate a significant increase in proline concentration as water stress levels increased, with an increase of 155.26% at 30% field capacity compared to 80% field capacity. Concerning the varieties, the statistical analysis results indicated there are major variances between the two mung bean kinds, and the Mexican variant. superior in proline content than the Iraqi variety. As for the two-way interaction between varieties and drought, The Mexican type has the highest proline content, according to the findings grown at 30% field capacity compared to the other treatments.The results of the three-way interaction (variety × concentration × drought) showed a significant reduction in the Iraqi variety treated with 100 parts per million and grown at 80% field capacity compared to the other treatments.

Table 3: Influence of Various ZnO Nanoparticle Concentrations on Proline Concentration (Micromoles/gram of fresh weight) in o Water Stressed Mung Bean

2.2 Peroxidase Enzyme Activity (POD)

POD enzyme activity was significantly reduced by spraying several amounts of ZnO nanoparticles, according to the statistical analysis results displayed in Table 4. With a drop of 20.25% in comparison to the control treatment, the concentration of 100 parts per million showed the largest decrease. POD enzyme activity increased significantly in response to decreasing moisture levels, increasing by 37.288% at 30% field capacity compared to mung bean plants growing at 80% field capacity.

In terms of the variations' effects, the POD enzyme activity of the Iraqi variety was much lower than that of the Mexican type. When it comes to the connection between concentration and types, the Mexican variety showed a much higher POD enzyme activity in plants that were not given ZnO nanoparticle treatment. For the three-way interaction (varieties × concentration × drought), the Iraqi variety treated with 100 parts per million of ZnO nanoparticles and grown at 80% field capacity recorded the lowest POD enzyme activity compared to other treatments.

Table 4 : The Impact of different Various ZnO Nanoparticle Concentrations on Peroxidase Enzyme Activity (Micromolar/mL) in Two Mung Bean Varieties Grown at Various Field Capacity Level

2.3 Catalase Enzyme Activity (CAT)

It is evident from Table 5, results that applying varying quantities of zinc oxide nanoparticles to plants significantly decreased the activity of the catalase (CAT) enzyme. In comparison to untreated plants, concentrations of 100 and 200 parts per million showed drops of 4.49% and 2.43%, respectively. In relation to moisture levels, plants at 80% field capacity showed a substantial drop in CAT enzyme activity, with a loss of 2.278% when compared to plants at 30% field capacity. 

As for the impact of the varieties, the results demonstrate a notable rise in CAT enzyme activity within the Mexican variety in contrast to the Iraqi variety. Regarding the interaction effect between concentration and drought, untreated plants showed higher CAT activity compared to treated plants exposed to the highest levels of water stress at 30% field capacity. In terms of the three-way interaction, The greatest value was reported for the Mexican variety in untreated plants grown at the moisture level of 30% field capacity.

Table 5 : Influence of Applying Various Zinc Oxide Concentrations on Catalase Enzyme Activity (Micromolar/mL) in Two Mung Bean Varieties Grown at Various Field Capacity Levels

 3-Estimation of Carbohydrate Content

The results in Table 6 indicate that treating mung bean plants with zinc oxide nanoparticles at concentrations of 100 and 200 parts per million led to a significant decrease in carbohydrate content. The reduction was particularly noticeable, with decreases of 12.20% and 4.65% respectively, compared to untreated plants.

Regarding the impact of moisture levels, there was a significant increase in carbohydrate content at 30% field capacity, with increases of 7.97% and 18.91% compared to the 50% and 80% field capacity treatments, respectively. The effect of the varieties shown that the Mexican type had a greater carbohydrate content than the Iraqi variety.

For the interaction between varieties and concentration, The Mexican variant has a larger carbohydrate content than the Iraqi variety in untreated plants compared to plants treated with nanoparticles. The three-way interaction (varieties × concentration × drought) indicated a significant decrease in carbohydrate content in the Iraqi variety treated with 100 parts per million of zinc oxide nanoparticles and grown at 80% moisture level.

Table 6: The Impact of Application Various Concentrations of ZnO-NPs on the Carbohydrate Content of Mung Beans Grown under Water Stress Environments

One kind of abiotic environmental stress that can result from a number of variables, including soil salinity, drought, and high temperatures, is water stress, also known as water shortage. Plant growth and production are greatly impacted by this [21].resulting in significant financial losses for the agriculture industry[22]. Tables (1,2) show that the mung bean plants under water stress had a decreased chlorophyll content (chlorophyll a, b) in their leaves at a field capacity of 30%, suggesting the most severe stress in comparison to the control plants. This reduction might be explained by the detrimental effects of water stress on physiological and biochemical processes in plants, which in turn affect plant pigments. Water shortage physiologically causes a drop in the relative water content of leaves, which lowers the amount of chlorophyll. Biochemically, water stress causes a decrease in soluble protein concentration and an increase in reactive oxygen species (ROS) and malondialdehyde formation [23]. Furthermore, as tables (4,5) demonstrate, water stress increases the activity of antioxidant defense enzymes including catalase (CAT) and peroxidase (POD). Plant development and photosynthesis are impacted as a result of the decrease in pigments caused by these physiological and biochemical changes. This is supported by [24], who found that removing almost 70% of the water from the soil had observable detrimental impacts, such as a marked reduction in the amount of color that wheat plants contained.

Table (6) illustrates how field capacity affects the amount of carbohydrates present. At the lowest field capacity of 30%, there is a notable rise in the amount of carbohydrates present in the leaf tissue. The decrease in starch content in the plant's leaves, stems, and roots may be the cause of the buildup of carbohydrates during water stress. Additionally, in response to a water deficit, levels of total fructans and soluble sugars rise. In order to maintain osmotic balance within the cell and shield plants from dryness, carbohydrates are essential [25]. This is consistent with research by [26], which shows that plants under water stress try to store carbohydrates, which function as osmolytes to assist the plant survive dry spells, in an effort to maintain the turgor pressure inside their cells. 

Regarding Table (3), findings, which show a considerable rise in the content of the amino acid proline in mung bean plants under water stress, the buildup of proline in plant tissues is thought to be one of the primary markers of how water stress affects plants. This is likely due to the functions of proline in cells, as it helps maintain osmotic pressure balance within plant cells. It has been observed that when proline accumulates in the cytoplasm, osmotic pressure decreases, thereby protecting cells from dehydration[27]. Additionally, proline serves as an antioxidant, protecting cell membranes and proteins from oxidative damage caused by drought. It also aids in maintaining the tertiary structure of proteins and stabilizing enzymes, thereby strengthening the plant's resistance to water stress. Additionally, proline controls the genes that control the plant's stress response. These results align with those of [28], who observed elevated proline levels in water-stressed fenugreek plants.

Notechnology is one of the most revolutionary disciplines of the twenty-first century.  (NPs) can be organic or inorganic, and they have gained popularity worldwide in recent years.  (ZnO-NPs) are among of the most widely utilized nanoparticles. ZnO-NPs are deemed physiologically safe for living creatures, and earlier research investigations have shown that they may boost plant growth and seed germination, as well as fight illnesses and protect plants through antibacterial activity[29]. Tables (1,2) reveal that the concentration of plant pigments increased in the presence of ZnO nanoparticles, particularly at 100 ppm. This rise might be attributable to ZnO NPs  ability to minimize drought-related damage, such as the generation of reactive oxygen species (ROS) and the reduction in chlorophyll pigments (a and b). They also improved plant growth nutrients because they promote chloroplast formation and function. Zinc promotes plant development by increasing photosynthesis and enzymatic activity [30]. Furthermore, ZnO NPs alter cell wall components and enhance cell membrane permeability, which leads to cell proliferation [31].The usage of ZnO-NPs increases the activity of the Rubisco enzyme, which is directly related to photosynthetic activity [32]. These findings are consistent with those of [33], who discovered that spraying eggplant plants with ZnO nanoparticles greatly decreased chlorophyll consumption while increasing photosynthetic activity during water stress. 

The benefits of zinc may be the cause of the leaves  increased chlorophyll concentration on plant growth and photosynthesis. Studies have shown that ZnO nanoparticles boost chlorophyll content, which in turn improves physiological growth traits and antioxidant properties in plants like mustard and tomatoes[34]. This increase in chlorophyll is crucial for photosynthesis, ultimately helping to promote the development of plants and productivity. Therefore the application of ZnO nanoparticles serves as a catalyst to boost chlorophyll levels in the leaves, promoting overall plant health and performance[35]. These results are consistent with findings by[36], which indicate that the use of ZnO nanoparticles increases total chlorophyll levels, establishing a direct relationship between zinc and chlorophyll production.

The study's findings, which are displayed in Tables (4,5), suggest that the antioxidant enzymes catalase (CAT) and peroxidase (POD) were less active when ZnO nanoparticles were sprayed on mung bean leave .This decrease can result from zinc ions' inhibitory action on these enzymes. Studies have shown that exposure to increasing concentrations of zinc ions results in a rapid decrease in peroxidase activity[37]. Additionally, the reduction in CAT and POD enzymes when using ZnO nanoparticles might be attributed to the specific effects of these particles on enzymes involved in redox reactions[34]. These findings contrast with those of[38], who found that the action of catalase and Peroxidase levels rose. when ZnO nanoparticles were used on mung beans subjected to heat stress. Non-enzymatic antioxidants play a crucial role when plants are subjected to any type of stress. As observed in Table(3), the levels of proline increased in plant tissues under water stress. However, when the vegetative parts were sprayed with zinc oxide nanoparticles, the levels of the amino acid proline decreased. This reduction might be due to the nanoparticles acting as a defense mechanism for the plant when exposed to any kind of biotic or abiotic stress. Therefore, the decrease in proline levels in mung bean leaves could indicate a change in the plant's response mechanisms to stress, likely due to the enhanced defense mechanisms provided by the nanoparticles[39]. Our findings are consistent with a study by[40], which also observed a decrease in proline levels in mung beans treated with ZnO nanoparticles under water stress conditions. he study's results indicate a decrease in carbohydrate levels in the leaf tissues of plants treated with ZnO nanoparticles at levels of 100 and 200 parts per million, as shown in Table (6). This outcome may be due to metabolic changes in the plants, which in turn lead to a reduction in carbohydrate accumulation[41]. These findings align with those of[42], who noted a notable decline in carbohydrate content in the leaves of Abelmoschus esculentus.   

The results of the study indicate:Exposing mung bean plants to different levels of water stress caused various responses depending on the treatments. The 80% field capacity was the optimal moisture level for the growth of both mung bean varieties (Iraqi and Mexican).The Mexican mung bean variety outperformed the Iraqi variety in most of the studied traits, indicating that it is more tolerant to water stress conditions.Spraying the vegetative groups of mung bean plants with zinc oxide nanoparticles had a positive impact on every attribute examined, providing the plants with the ability to withstand drought. The concentration of 100 parts per million was more effective than the other concentrations.The interaction between the two factors (zinc oxide nanoparticles and field capacity levels) had a positive effect, contributing to the improvement of most of the studied traits affected by water stress.

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