Vanadium Stress Alters Sweet Potato (Ipomoea batatas L.) Growth, ROS Accumulation, Antioxidant Defense System, Stomatal Traits, and Vanadium Uptake

Vanadium (V) is a heavy metal found in trace amounts in many plants and widely distributed in the soil. This study investigated the effects of vanadium concentrations on sweet potato growth, biomass, root morphology, photosynthesis, photosynthetic assimilation, antioxidant defense system, stomatal traits, and V accumulation. Sweet potato plants were grown hydroponically and treated with five levels of V (0, 10, 25, 50, and 75 mg L−1). After 7 days of treatment, V content at low concentration (10 mg L−1) enhanced the plant growth and biomass; in contrast, drastic effects were observed at 25, 50, and 75 mg L−1. Higher V concentrations negatively affect the relative water content, photosynthetic assimilation, photosynthesis, and root growth and reduce tolerance indices. The stomatal traits of sweet potato, such as stomatal length, width, pore length, and pore width, were also decreased under higher V application. Furthermore, V concentration and uptake in the roots were higher than in the shoots. In the same way, reactive oxygen species (ROS) production (hydrogen peroxide), lipid peroxidation (malondialdehyde), osmolytes, glutathione, and enzymes (catalase and superoxide dismutase) activities were increased significantly under V stress. In conclusion, V at a low level (10 mg L−1) enhanced sweet potato growth, and a higher level of V treatment (25, 50, and 75 mg L−1) had a deleterious impact on the growth, physiology, and biochemical mechanisms, as well as stomatal traits of sweet potato.


Introduction
Vanadium (V) is world's fifth most abundant transition element and deposit, mainly in China, the USA, Russia, and South Africa [1,2]. China is the leading producer and consumer of V, with 57% of V production globally. Around 26.5% of V-contaminated soil is present in southwest China [3,4]. V is widely distributed and mobilized in the surrounding environments by several natural events and anthropogenic activities, including weathering of parental rocks, redox processes, leaching, fertilizers usage, combustion, and industrial wastes, which as a result, contaminates the water, soil, and atmosphere [4,5]. V is deposited naturally in the soil in different mineral forms, and the average concentration of V ranges from 3 to 310 mg kg −1 in soil. The average V concentration in fresh, ground, and drinkable water is 0.5 µg L −1 , with peak concentrations in volcanic areas reaching 127.4 µg L −1 [6]. Accumulating V in the natural habitat increases soil and water contamination, ultimately affecting human health by causing nausea, vomiting, dizziness, and more seriously, leading to kidney damage [7]. Different studies have reported that V has detrimental effects on the

Seedling Collection, Growth Conditions, and Experimental Design
In this experiment, we used the "Haida HD7791" sweet potato cultivar. For the disinfection of sweet potato cuttings, 1 g L −1 carbendazim was used for 5-8 min. Afterwards, the sweet potato cuttings were kept in Ro water until the roots appeared. For the acclimation, the cuttings of sweet potato were grown in half Hoagland media (pH 5.8 ± 0.2). A hydroponic experiment in a controlled environment (25)(26)(27) • C for 16 h of photoperiod) was conducted to assess V's effect on sweet potato plants. For proper nutrient availability, the nutrient media were replaced after every 5 to 6 days. Subsequently, healthy and uniform seedlings were distributed among the five treatment groups. V (0, 10, 25, 50, and 75 mg/L) was applied as ammonium metavanadate (NH 4 VO 3 ). For the analysis of morphological and physiological measurements, samples were collected after 7 days of treatment with V or normal growth conditions and immediately transferred into a liquid nitrogen tank.

Growth Variables
Three independent seedlings were used to measure plant height (PH), the leaf area (LA), number of leaves (LN), and fresh and dry weights of the roots and shoots. A portable laser leaf area meter (CI-202) was used to measure the LA (topmost leaves). A ruler was used to measure the height of the seedlings. The seedlings were cut, and the fresh weight of the shoot and root was recorded. For recording the dry weight (DW), the samples were first dried at 105 • C for 30 min and then kept for drying at 70 • C for 3 days [32]. The plant's shoot DW susceptibility index (SDSI) was calculated as follows: Similarly, the following formula was used to calculate the plant's RDSI; RDSI = Root DW (stressed plants) Root DW (controlled plants) × 100 (2)

Relative Water Content Analysis
After recording the FW, the sweet potato leaves were immersed in ddH 2 O for four hours. After 4 h the leaves were weighed to determine the turgor weight (TW). To determine the dry weight (DW), the leaves were oven-dried for one day at 70 • C [32]. Finally, the RWC was measured using the following formula:

Root Morphology
After surface rinsing, the roots were washed with ddH 2 O and scanned with the Imagery Screen (Epson Expression 11000XL, Regent Instruments, Chemin Sainte-Foy, QC, Canada) to observe different root traits. The images obtained from the root scanner were analyzed with the WinRHIZO 2003a software program [13].

Gas Exchange Parameters
Gas exchange parameters of sweet potato leaves were determined for completely matured leaves utilizing a portable photosynthesis system (CIRAS-3, Hansatech Co., Amesbury, MA, USA) [13].

V Determination, Uptake, and Translocation
A super microwave-assessed digestion system (Anton Paar, Multiwave 7000, Styria, Garz, Austria) was used to digest plant samples (100 mg dry weight) with 2 mL HNO 3 , 0.5 mL H 2 O 2 , and 1 mL deionized water. We used the standard reference material (GBW10015) in triplicate for quality control and assurance (obtained from the Chinese Academy of Geological Sciences, Langfang, China). The reference CRM standard value was V(10 −6 ) 0.87 ± 0.23. The working standards of V (0-200 µg/L) were made using a standard stock solution (GSB04-1759-2004, Beijing, China) containing 1000 mg/L of V. The inductively coupled plasma mass spectrometer (ICP-MS) (Perkin Elmer, NexION 5000, Waltham, MA, USA) was used to measure the V content [32]. The detection limit (DL) of standard V in solution was 0.01 µg/L (0.001522 ppb), the correlation coefficient was 0.999977, and the recovery of CRM standard V ranged from 86.5% to 93%. The subsequent formula to measure V uptake and translocation was used: V uptake (mg) = V concentrations in the tissues × dry weight of the tissues (4) Translocation = V concentration in the plant shoots V concentration in the plant roots (5)

Measurement of Photosynthetic Pigments
Leaf samples (0.1 g FW) were mixed with 80% acetone, followed by centrifugation for 15 min at 8000× g. A microplate reader (Infinite M200 PRO, TECAN, Männedorf, Swiss) was used for measuring the absorbance of chlorophyll a (chl a), chl b, and carotenoids (Car) at 663, 646, and 470 nm, respectively. Finally, the concentration of chlorophyll was calculated with the subsequent formula reported by Kumar et al. [16].

Determination of Malondialdehyde (MDA)
A kit (A003-1-1) was used to quantify MDA following the thiobarbituric acid (TBA) method. A glass homogenizer was used to properly homogenize 100 mg of fresh leaves in 900 µL of extraction buffer provided by the company. The homogenate was centrifuged at 5000× g for 15 min, followed by three centrifugations of 15 s each at 4000× g, with a 30 s interval between each centrifugation, with the final centrifugation of 3500× g for 10 min. After that, the supernatant was collected and mixed with the working fluid (combination of R1: clarifying agent, R2: buffering agent, and R3: color developer in the ratio of 0.1:3:1) provided by the company, then the mixture was boiled at 95 • C for 20 min. After cooling, the absorbance at 530 nm was determined using a full-wavelength microplate reader (Infinite M200 PRO, TECAN, Männedorf, Swiss) [32].

Determination of Proline and Soluble Sugars
Proline was calculated by using an assay kit (A107-1-1). Fresh leaf samples were homogenized with buffer available in the kit and tested at 520 nm following the company's protocol. Approximately 50 mg of fresh leaves were homogenized in 0.45 mL ddH 2 O for the analysis of soluble sugars. The homogenate was boiled at 95 • C for 15 min and then centrifuged at 7500× g for 15 min. After that, the supernatant was collected and diluted with ddH 2 O at 1:9. Using a test kit (A145-1-1), the soluble sugar content of the diluted extracts was determined at 620 nm [17,32].

Determination of Total Polyphenols and Flavonoid Content
A fresh leaf sample of 1 g was homogenized with 60% ethanol. After that, 1.25 mL of 10% Folin-Ciocalteu reagent was added to 250 µL of extract and 1 mL of sodium carbonate (0.75 g/mL). After incubating for 15 min at 45 • C, the mixture was allowed to remain at Antioxidants 2022, 11, 2407 5 of 20 room temperature for 30 min. As a final step, the absorbance was recorded at 765 nm, and the results were presented as Gallic acid equivalents per gram (GAE/g) to quantify total polyphenols [16,34].
Approximately 0.25 mL of NaNO 2 solution (0.5 g/mL) and 2 mL ddH 2 O were mixed with 0.5 mL of extract to measure flavonoids. After being retained at 25-28 • C for 5 min, 150 µL of aluminium chloride (1 g/mL), 1 mL of NaOH (1 M), and 1.2 mL of ddH 2 O were added simultaneously. As a final step, its absorbance was measured at 510 nm with Catechin (CAE) used as a standard, and its results were presented as CAE/g [16,34].

Scanning Electron Microscopy (SEM)
To observe the stomatal morphology, we used a published protocol [30]. To remove any debris, leaves were acetylated in 80% ethanol for two to three min. The tiny sections of leaf were prepared using s-cutting, and after that platinum was used to fix the abaxial and adaxial surfaces and sputtered using Leica Mikrosystem GmbH (ACE600) for 25 min, and finally examined under a SEM (Thermo Scientific, Verios G4 UC, Waltham, MA, USA).

Statistical Analysis
Three individual replications were used to obtain phenotypic, physiological, and biochemical indices. Significant differences (p ≤ 0.05) between means were determined using SPSS 25.0 software, and Duncan tests were applied for the means comparison, while ± represents a standard error (S.E). Figures were plotted with GraphPad Prism 7. The "ggplot2" package in R (version 3.3.4, https://CRAN.R-project.org/package=ggplot2 (accessed on 24 August 2022)) was used for principal component analysis (PCA) and Pearson correlation analysis.

Leaf Gas Exchange Elements
This study showed that gas exchange elements were enhanced by the application of 10 mg L −1 V treatment; in contrast, a higher application of V (25, 50, and 75 mg L −1 ) showed a negative effect on the gas exchange elements of sweet potato leaves in comparison to the control plant ( Figure 3). Comparing the 10 mg L −1 V-treated group to the control group, the transpiration rate (Tr), photosynthesis rate (Pn), stomatal conductance (Gs), and intercellular CO 2 (Ci) increased by 10.6%, 23.9%, 33.2%, and 0.8%, respectively ( Figure 3). Conversely, maximum reductions of 65.4%, 76.5%, 61.8%, and 62.8% were noticed in the Tr, Pn, Gs, and Ci, respectively when compared to the control ( Figure 3).

Concentration, Uptake, and Translocation of Vanadium
This study depicted that V treatment significantly raised the V concentration in both shoots and roots, and maximum concentrations were recorded in 75 mg L −1 treated plants; 32.03 mg kg −1 DW in the shoots of sweet potato and 52.68 mg kg −1 DW in the roots of sweet potato (Table 3). Similarly, the accumulation of V was found to be higher in the roots than in the shoots. Moreover, V uptake by shoots and roots of sweet potato significantly augmented as the level of V increased. In the same way, the translocation of V from root to shoot was also raised significantly by the increment of V concentration (Table 3).  10 mg L V treatment; in contrast, a higher application of V (25, 50, and 75 mg L ) showed a negative effect on the gas exchange elements of sweet potato leaves in comparison to the control plant ( Figure 3). Comparing the 10 mg L −1 V-treated group to the control group, the transpiration rate (Tr), photosynthesis rate (Pn), stomatal conductance (Gs), and intercellular CO2 (Ci) increased by 10.6%, 23.9%, 33.2%, and 0.8%, respectively ( Figure 3). Conversely, maximum reductions of 65.4%, 76.5%, 61.8%, and 62.8% were noticed in the Tr, Pn, Gs, and Ci, respectively when compared to the control ( Figure 3).

Concentration, Uptake, and Translocation of Vanadium
This study depicted that V treatment significantly raised the V concentration in both shoots and roots, and maximum concentrations were recorded in 75 mg L −1 treated plants; 32.03 mg kg −1 DW in the shoots of sweet potato and 52.68 mg kg −1 DW in the roots of sweet potato (Table 3). Similarly, the accumulation of V was found to be higher in the roots than in the shoots. Moreover, V uptake by shoots and roots of sweet potato significantly augmented as the level of V increased. In the same way, the translocation of V from root to shoot was also raised significantly by the increment of V concentration (Table 3).

Photosynthetic Pigments
The chlorophyll (Chl) was significantly influenced by the application of V stress. Compared to the control, adding 10 mg L −1 V did not significantly raise the concentration of chlorophyll and carotenoid (Car) (Figure 4). On the other hand, higher treatment of V (25, 50, and 75 mg L −1 ) showed a significant negative impact on the photosynthetic pigments, and utmost reduction was detected at 75 mg L −1 . Compared to the control, 77% reduction in the content of total chl, 65.9% in chl a, 70.5% in chl b, and 50.3% in Car were detected ( Figure 4).
The chlorophyll (Chl) was significantly influenced by the application of V stress. Compared to the control, adding 10 mg L −1 V did not significantly raise the concentration of chlorophyll and carotenoid (Car) (Figure 4). On the other hand, higher treatment of V (25, 50, and 75 mg L −1 ) showed a significant negative impact on the photosynthetic pigments, and utmost reduction was detected at 75 mg L −1 . Compared to the control, 77% reduction in the content of total chl, 65.9% in chl a, 70.5% in chl b, and 50.3% in Car were detected ( Figure 4).

Lipid Peroxidation (MDA) and Reactive Oxygen Species (H 2 O 2 ) Content
The V treatment considerably provoked MDA and H 2 O 2 levels in the leaves (p < 0.05; Figure 5A,B). The rise of V treatment caused an increase in MDA and H 2 O 2 content; the highest MDA and H 2 O 2 content was present in 75 mg L −1 V treatment compared to the control. The MDA content in 75 mg L −1 V was 928.9% higher than the control ( Figure 5A), where the H 2 O 2 content was 665% higher ( Figure 5B).

Lipid Peroxidation (MDA) and Reactive Oxygen Species (H2O2) Content
The V treatment considerably provoked MDA and H2O2 levels in the leaves ( Figure 5A,B). The rise of V treatment caused an increase in MDA and H2O2 cont highest MDA and H2O2 content was present in 75 mg L −1 V treatment compare control. The MDA content in 75 mg L −1 V was 928.9% higher than the control (Figu where the H2O2 content was 665% higher ( Figure 5B).

Osmolytes Production
The proline content was significantly increased as the level of V increased (p The maximum rise of 448.3% in proline content was detected at 75 mg L −1 V tre

Osmolytes Production
The proline content was significantly increased as the level of V increased (p < 0.05). The maximum rise of 448.3% in proline content was detected at 75 mg L −1 V treatment ( Figure 6A). Soluble sugars were also significantly higher in V-treated plants (p < 0.05). The content of soluble sugars in the leaves of sweet potato was increased to 50 mg L −1 ; however, at 75 mg L −1 V, the content of soluble sugars decreased but was still significantly higher (97.6%) than the control plants ( Figure 6B). Furthermore, the results exhibited that the total proteins were significantly increased at 10 mg L −1 V treatment; later, a significant decrease was observed at a higher level of V treatment (p < 0.05). The lowest protein content (0.029 mg g −1 ) was observed at 75 mg L −1 V treatment compared to the control ( Figure 6C).

Antioxidants
The GSH content in the leaf of sweet potato increased with the rise of V treatment, and the maximum concentration was observed at 75 mg L −1 V treatment ( Figure 7A). The GSH content at 10 and 25 mg L −1 V treatment was insignificantly increased (16.4 and 28.5%); however, a significant increase was observed at 50 and 75 mg L −1 V treatment, which were 134% and 324% higher than the control, respectively. Total polyphenols and flavonoid concentrations decreased significantly with the rise of V treatment (p < 0.05). Interestingly, total polyphenols and flavonoids drastically reduced at 10 and 25 mg L −1 V treatment, then again increased at 50 and 75 mg L −1 V treatment, however still significantly lower than in the control plants ( Figure 7B,C).

Antioxidants
The GSH content in the leaf of sweet potato increased with the rise of V treatment, and the maximum concentration was observed at 75 mg L −1 V treatment ( Figure 7A). The GSH content at 10 and 25 mg L −1 V treatment was insignificantly increased (16.4 and 28.5%); however, a significant increase was observed at 50 and 75 mg L −1 V treatment, which were 134% and 324% higher than the control, respectively. Total polyphenols and flavonoid concentrations decreased significantly with the rise of V treatment (p < 0.05). Interestingly, total polyphenols and flavonoids drastically reduced at 10 and 25 mg L −1 V treatment, then again increased at 50 and 75 mg L −1 V treatment, however still significantly lower than in the control plants ( Figure 7B,C).

Antioxidant Enzymes
Antioxidant enzyme activities were significantly influenced by V treatment (p < 0.05). We found a significant decrease in APX and POD activities with the rise of V concentration (p < 0.05; Figure 8A,D), and maximum reduction was detected at 75 mg L −1 of V treatment. As compared to the control, a 62.1% reduction in APX and a 57.5% in POD was detected at 75 mg L −1 V treatment. Conversely, the CAT and SOD were positively influenced by V treatment, and a significant increase was observed with the rise in V concentration (p < 0.05; Figure 8B,C). Furthermore, the highest activities of CAT and SOD were observed at the 75 mg L −1 V treatment, and a 1085% increase in CAT and a 164.7% increase in SOD were observed compared with the control.

Antioxidant Enzymes
Antioxidant enzyme activities were significantly influenced by V treatment (p < 0.05). We found a significant decrease in APX and POD activities with the rise of V concentration (p < 0.05; Figure 8A,D), and maximum reduction was detected at 75 mg L −1 of V treatment. As compared to the control, a 62.1% reduction in APX and a 57.5% in POD was detected at 75 mg L −1 V treatment. Conversely, the CAT and SOD were positively influenced by V treatment, and a significant increase was observed with the rise in V concentration (p < 0.05; Figure 8B,C). Furthermore, the highest activities of CAT and SOD were observed at the 75 mg L −1 V treatment, and a 1085% increase in CAT and a 164.7% increase in SOD were observed compared with the control.

Effects on Leaf Morphology
The present work also attempted to study the effects of V treatment on leaf morphology under SEM. The results showed that leaf morphology under 10 mg L −1 V treatment was statistically the same as the control plants ( Figure 9). Compared to the control plants, stomatal length, width, pore length, and pore width were increased by 4.7%, 2.6%, 10.1%, and 10.1%, respectively, under 10 mg L −1 V treatment (Table 4). In contrast, stomata size was significantly affected under a high level of V treatment (25, 50, and 75 mg L −1 ), and the maximum deleterious effects were observed at 75 mg L −1 V treatment (Figure 9). Compared to the control leaf, stomata length under 75 mg L −1 V treatment was reduced by 47.2%; likewise, the width of the stomata was decreased by 80.1%, pore length by 71%, and pore width by 87.1%. Closed and small stomata were observed due to stress conditions, which showed small stomatal openings. The result indicated that V induced stomatal closing and reduced its size. Antioxidants 2022, 11, x FOR PEER REVIEW 13 of 22

Effects on Leaf Morphology
The present work also attempted to study the effects of V treatment on leaf morphology under SEM. The results showed that leaf morphology under 10 mg L −1 V treatment was statistically the same as the control plants ( Figure 9). Compared to the control plants, stomatal length, width, pore length, and pore width were increased by 4.7%, 2.6%, 10.1%, and 10.1%, respectively, under 10 mg L −1 V treatment (Table 4). In contrast, stomata size was significantly affected under a high level of V treatment (25, 50, and 75 mg L −1 ), and the maximum deleterious effects were observed at 75 mg L −1 V treatment ( Figure 9). Compared to the control leaf, stomata length under 75 mg L −1 V treatment was reduced by 47.2%; likewise, the width of the stomata was decreased by 80.1%, pore length by 71%, and pore width by 87.1%. Closed and small stomata were observed due to stress conditions, which showed small stomatal openings. The result indicated that V induced stomatal closing and reduced its size.

Pearson's Correlation and Heat-map Analysis
The negative correlation between physiological parameters and osmolytes, GSH, CAT, and SOD in sweet potato plants treated with different concentrations of V demonstrates the significant reduction in the plant's phenotypic and physiological traits ( Figure 10). All plant phenotypic parameters and root and shoot concentrations of V were negatively correlated, showing that V treatment adversely affected plant growth and development. Similarly, osmolytes, GSH, CAT, SOD, and V concentrations and uptake in sweet potato showed a negative correlation with photosynthetic pigments, assimilation, and stomatal traits ( Figure 10). However, the phenotypic parameters showed a positive correlation with photosynthetic pigments and assimilation, indicating that plants can grow larger and produce more biomass at a high rate of photosynthetic pigments. A heatmap-histogram analysis of different growth traits of sweet potato under different levels of V treatment was also constructed (Figure 11). A significant difference was observed with different colors in the different V treatments and responses of different physiological and biochemical parameters, as well as V concentration and uptake. However, traits with red color indicate insignificant differences within the V treatments. This heatmap-histogram showed a noticeable difference among the growth traits and uptake of V in the sweet potato plant. , Gs (stomatal conductance), Chl (total chlorophyll), and Tr (transpiration rate). Measured determinants and strength correlation matrix. The color code for Pearson correlation coefficients (r), with r = 2, r = 0, and r = −2 denoting red, orange, green, and gray, respectively. A stronger association is denoted by better anisotropy, and the slope of the corresponding line or ellipse represents the trend of that association (positive or negative). Additionally, the ellipses' direction and anisotropy indicate the slope and strength of the relationship. According to the first principal component order, variables were arranged.
A heatmap-histogram analysis of different growth traits of sweet potato under different levels of V treatment was also constructed (Figure 11). A significant difference was observed with different colors in the different V treatments and responses of different physiological and biochemical parameters, as well as V concentration and uptake. However, traits with red color indicate insignificant differences within the V treatments. This heatmap-histogram showed a noticeable difference among the growth traits and uptake of V in the sweet potato plant.

Discussion
Globally, agricultural soil has been polluted with several kinds of soil pollutants with anthropogenic activities. Several studies have indicated that heavy metals, such as Cd, Ni, Pb, and V, are the primary cause of soil pollution. V used in the steel industries and accumulation in the agricultural land and water gained consideration by researchers in recent years [35][36][37]. V accumulation showed a deleterious effect on living organisms, including plants, animals, and humans. In order to better understand the mechanisms underlying V toxicity in sweet potato, we investigated the phenotypic, physiological, and biochemical processes under various levels of V treatments. Plant growth and biomass are not only used to study different types of heavy metal stress, but also used to evaluate the tolerance level of plants against them. Heavy metals cause plant toxicity and negatively affect the plant length and fresh and dry biomass [38]. However, its toxicity varies with the plant species, chemical structure and formula, concentration, and recurrence of use [39]. However, at higher concentrations, V severely inhibits plant growth and development [40]. In the present study, we observed an improvement in the growth of sweet potato under 10 mg L −1 V treatment compared to the control plant (Figure 1). According to some studies, plants treated with V had increased height, growth, and fresh and dry biomass [11,12]. Aihemaiti et al. stated that plants generate more biomass at low levels of V due to enhanced chlorophyll biosynthesis [22]. However, many studies reported a decrease in the plant height, growth, and fresh biomass under a higher level of V stress [38,41], and the

Discussion
Globally, agricultural soil has been polluted with several kinds of soil pollutants with anthropogenic activities. Several studies have indicated that heavy metals, such as Cd, Ni, Pb, and V, are the primary cause of soil pollution. V used in the steel industries and accumulation in the agricultural land and water gained consideration by researchers in recent years [35][36][37]. V accumulation showed a deleterious effect on living organisms, including plants, animals, and humans. In order to better understand the mechanisms underlying V toxicity in sweet potato, we investigated the phenotypic, physiological, and biochemical processes under various levels of V treatments. Plant growth and biomass are not only used to study different types of heavy metal stress, but also used to evaluate the tolerance level of plants against them. Heavy metals cause plant toxicity and negatively affect the plant length and fresh and dry biomass [38]. However, its toxicity varies with the plant species, chemical structure and formula, concentration, and recurrence of use [39]. However, at higher concentrations, V severely inhibits plant growth and development [40]. In the present study, we observed an improvement in the growth of sweet potato under 10 mg L −1 V treatment compared to the control plant ( Figure 1). According to some studies, plants treated with V had increased height, growth, and fresh and dry biomass [11,12]. Aihemaiti et al. stated that plants generate more biomass at low levels of V due to enhanced chlorophyll biosynthesis [22]. However, many studies reported a decrease in the plant height, growth, and fresh biomass under a higher level of V stress [38,41], and the impact varies from organ to organ of the plants [8]. Similarly, the current study depicted that 25, 50, and 75 mg L −1 V treatment considerably reduced the growth of sweet potato, and maximum reduction was observed at 75 mg L −1 (Tables 1 and 2). The SDSI and RDSI increased at 10 mg L −1 ; in contrast, a negative correlation was observed with a further increase of the V level (25,50, and 75 mg L −1 ) in the growth medium. A higher level of V stress initiated an ionic imbalance and interrupted their function in metabolic pathways, which eventually affected the process of growth and development of the plant. RWC is a simple and reliable parameter for calculating plant stress. Under various V treatments, we found a decrease in the RWC, indicating that the sweet potato plants were under stress. Osmotic adjustment is impaired in sensitive plant species. A previous study on lettuce also showed a decrease in water content with an increase of V concentration [42]. Under heavy metal stress, various studies also reported a reduction in RWC in the leaves of barley and maize [43,44]. The present study showed that a low level of V treatment (10 mg L −1 ) induced the root traits of sweet potato ( Figure 2). Variations in root size and morphology can improve ionic uptake and translocation, ultimately enhancing plant growth and development [45]. A previous study also reported the expansion of root traits under low V treatment (10 mg L −1 ) [11]. In contrast, a higher level of V (25, 50, and 75 mg L −1 ) caused a decrease in these root traits (Figure 2). At a higher level of V treatment, the plant produced comparatively shorter, less lateral, and coralloid structural roots. The decline in root traits under high V levels might have been due to the disruption in mitotic cell division, possibly hampering root tip development [46,47]. Previous studies also reported the decrease of root traits under a higher level of V treatment in watermelon, pepper, and rice [11,13,20]. In addition, V application significantly reduced root morphological traits in many plants [22]; these studies are in agreement with the findings of the present study.
Photosynthetic assimilation is the plant's ability to use CO 2 and perform many metabolic activities [48]. Heavy metal stress drastically affects photosynthesis and significantly affects carbohydrate synthesis. In the current study, we found an increase in these gas exchange elements under 10 mg L −1 treatment; however, a significant decrease in these gas exchange elements was observed at a higher level of V stress (Figure 3). A previous study reported a minor increase in the photosynthetic rate and intercellular CO 2 at a low level of V (10 mg L −1 ) treatment; however, a higher level of V showed a significant reduction in these gas exchange elements in pepper plants [11]. Similarly, another study revealed that V treatment in rice plants caused a reduction in gas exchange elements, which agrees with the current study [13]. At high concentrations, V imposes adverse effects on the physiological processes and photosynthetic assimilation, limiting energy production, and impairing biomass and development of plants [49,50]. V stress possibly reduces photosynthetic activities by affecting the chloroplast and ultrastructures and also disturbs their electron transport mechanisms.
The leaf stomata are responsible for regulating transpiration and CO 2 transport under adverse environmental conditions [51]. The plant experiences cytotoxicity due to the increased concentration of heavy metals, which affects ionic absorption, cell cycle arrest, lipid peroxidation, and ultimately results in cell death [52,53]. Heavy metal interaction with guard cells triggers stomatal closure [54]. V toxicity mediates stomatal abnormalities, such as a reduction in the stomatal length, width, pore length, and width of guard cells ( Figure 9 and Table 4). A study reported that the reduction in the stomata size is linked with more distorted stomata under an elevated level of heavy metals in the leaves [30,55]. Photosynthesis, transpiration, and gas exchange can be adversely affected by reductions in stomatal size and closure frequency. From the present study, it has been elucidated that increasing V level decreases stomatal size and closing stomata, which reflects a decrease in photosynthetic assimilation, transpiration rate, and gas exchange.
In the present study, the V concentration, uptake, and translocation had a positive correlation with the rise of V application, and the roots showed higher V content than the shoots ( Table 3). The concentration, uptake, and translocation of V to the aerial part are reduced by chelation and fixation of V with a polar compound, stimulation of calcium with a stable compound, and root and stem compartmentalization [56]. A previous study reported that tomato plants treated with V have more V in the roots than in the leaves [36]. In the same way, lettuce, tobacco, alfalfa, milkvetch root, and swamp morning glory treated with V also showed the same trend, and they have more V accumulation in roots than the leaves [1,10,35,42]. Generally, the increase in chlorophyll level characterizes plant photosynthesis assimilation and growth. The reduction in the chlorophyll content was found under different environmental stresses. A higher level of V treatment (25,50, and 75 mg L −1 ) reduced the T.Chl, Chl a, b, and carotenoid content (Figure 4). Similarly, previous studies revealed a significant reduction in the chlorophyll content under exposure to V stress in watermelon and pepper [11,13,20]. The reduction in photosynthetic pigments under V stress might be due to the variations in the membrane permeability and the devastation of the elements prompted by oxidative stress [57]. This increase in ROS production causes a reduction in chlorophyll content [15].
Lipids peroxidation is an indication of oxidative stress that is induced by different abiotic stress, and the higher MDA level also indicates cell membrane injury; it is a wellestablished sign for assessing plants introduced to metal stress [32]. The current study described that the V application considerably boosted the MDA content of sweet potato ( Figure 5A). Similarly, previous studies described that V-treated rice and pepper seedlings exhibited an increase in the MDA content [11,13]. Plants produce ROS due to the reaction of heavy metals and fatty acids [58]. H 2 O 2 is the main component of ROS and its production increases with heavy metal stress. The current study also reported that an increase in V stress in sweet potato causes a significant increment in H 2 O 2 level ( Figure 5B). Previous studies also observed a significant increment in the H 2 O 2 level when exposed to V stress in rice, tomato, and watermelon plants [13,20,36]. A review article by Chen et al. also highlighted that many articles followed the same pattern of increasing MDA and H 2 O 2 under V stress [59]. To deal with these circumstances, the plant possesses a defense mechanism, including osmolytes, antioxidants, and enzymes.
The reduction of osmolytes, such as proline, soluble sugars, and proteins are linked with the leaf water content, which could lead to cellular desiccation and osmotic stress in sweet potato ( Figure 6). According to the previous literature, plants exhibit a similar pattern by increasing proline and soluble sugars in response to increased cellular desiccation under V stress [6,11,22,42]. These osmolytes increase under abiotic stress conditions [17,32]. Moreover, these osmolytes might not only assist in protecting plant cellular membranes, but also help in maintaining turgor pressure, which minimizes the deleterious effect of vanadium toxicity. The total proteins are sensitive to heavy metal stress, and different studies reported a reduction of proteins with increased metal stress [13]. The present study showed a significant decrease in total protein content at a higher level of V treatment (25,50, and 75 mg L −1 ) ( Figure 7A). Heavy metal stress reduces the proteins by prompting toxic effects, damaging the ability of many enzymes with functional thiol groups [60]. The results of the present study agree with the reports of the Refs. [36,61], who stated that total protein content was reduced under V stress. The GSH can enhance the tolerance of plants under metal stress. Furthermore, GSH acts as a ROS scavenger, detoxifying the O 2 •− and HO • radicals [32]. This study depicted an increase of GSH under V stress ( Figure 7B). GSH levels increase under heavy metal stress due to increased activities of γ-glutamylcysteine synthetase and glutathione synthetase [62]. Our findings agree with previous reports, which described the increment in GSH content under heavy metal stress [61,63]. The secondary metabolites, such as polyphenols and flavonoids, not only enhance the enzymatic activities of the plants but also play a vital role as antioxidants in stress environments [17]. Izbiańska et al. and Kisa et al. reported a decrease in phenolics under heavy metal stress [64,65]. This study depicted that V-treated plants have a significant reduced level of polyphenols and flavonoids ( Figure 7C,D). This reduction in the polyphenols and flavonoids biosynthesis under V stress could be due to the decreased activity of essential enzymes of phenylpropanoid pathways [65,66]. Antioxidant enzymes are essential in reducing ROS production and oxidative stress under different environmental stress conditions [17,32]. In this study, we found a decrease in the activity of POD and APX with the rise in V level. In contrast, CAT and SOD significantly increased with the increase of V treatment (Figure 8). This increase in antioxidant enzyme activity may be attributed to the effect of the V ion on free-oxygen radical production. Tobacco plants treated with V have depicted an increment of CAT and SOD activities [10]. Likewise, watermelon and pepper plants under V stress have also depicted increased CAT and SOD activities [11,20]. The results of the current study also agree with studies on rice, chickpeas, oilseed, and tomato under V, Se, and Ni stress, respectively. [41,61,67,68]. Similarly, many other plants have also shown increased activities of these antioxidant enzymes under different levels of V treatments [59]. The antioxidant enzyme activities are powerful ROS scavengers and regulate the plant's cellular membranes under abiotic stress conditions.

Conclusions
In the present study, we found that 10 mg L −1 V treatment improved the growth and biomass of the sweet potato plant. However, V at higher levels (25,50, and 75 mg L −1 ) significantly reduced the growth of sweet potato by altering its physiological and biochemical mechanisms. The higher level of V treatments (25,50, and 75 mg L −1 ) significantly reduced the RWC, chlorophyll content, gas exchange elements of leaf, and stomatal traits, which eventually affected the growth of sweet potato. The decline in root morphological traits was observed at the higher V treatment, and we also detected maximum V concentration and uptake in the roots than in the shoots of sweet potato. It was also observed that the V application increased ROS production. Overall, this study will help to understand the physiological tolerance mechanism of V in sweet potato plants. In this study, we focused on the effect of V stress on the morphological and physiological aspects of sweet potato. Besides, this study was conducted in a hydroponically controlled environment, and thus, open-field experiments are encouraged to unravel the more precise effects of V on sweet potato, as in open-field conditions the plants are exposed to several other biotic and abiotic stresses. Moreover, further studies are also required to investigate the molecular mechanism underlying cell death induced by V toxicity in the sweet potato plant. Furthermore, we suggest using various chemicals, phytohormones, and nanoparticles to prevent V toxicity and stimulate sweet potato growth and production.

Data Availability Statement:
The data presented in this article will be made available without any reservation.