*3.10. 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−<sup>1</sup> 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−<sup>1</sup> 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−<sup>1</sup> V treatment (Figure 9). Compared to the control leaf, stomata length under 75 mg L−<sup>1</sup> 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.

**Figure 8.** Influence of various V treatments on the antioxidant enzymes activities in the leaves of sweet potato. (**A**) APX, (**B**) CAT, (**C**) SOD, and (**D**) POD. Duncan's test indicates a significant difference (*p* < 0.05) between the means of the five treatments indicated by different alphabetical letters.

**Figure 9.** Effect of different V treatments on the stomatal traits of sweet potato leaf. (**A**) Ck, (**B**) 10 mg L<sup>−</sup>1, (**C**) 25 mg L<sup>−</sup>1, (**D**) 50 mg L<sup>−</sup>1, and (**E**) 75 mg L<sup>−</sup>1.

#### *3.11. 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.

**Figure 10.** Pearson's correlation analysis (PCA) (*p* < 0.05) was calculated among different traits of V-treated sweet potato seedling. VSC (V shoot concentration), GSH (reduced glutathione), VSU (V shoot uptake), H2O2 (hydrogen peroxide), CAT (catalase), VRC (V root concentration), VRU (V root uptake in root), SOD (superoxide dismutase), MDA (malondialdehyde), SS (soluble sugars), Pro (proline), PP (total polyphenols), APX (ascorbate peroxidase), TF (total flavonoids), TP (total proteins), POD (peroxidase), Pn (photosynthetic assimilation), Car (carotenoids), Ci (Intercellular CO2), 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.


**Table 4.** Effect of different V treatments on the stomatal traits of sweet potato leaf.

Duncan's test specifies a significant difference (*p* < 0.05) between the means of the five treatments indicated by different alphabets.

**Figure 11.** Heatmap-histogram correlation between studied physiological and biochemical parameters under various V treatments in sweet potato.

#### **4. 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 [35–37]. 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−<sup>1</sup> 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−<sup>1</sup> V treatment considerably reduced the growth of sweet potato, and maximum reduction was observed at 75 mg L−<sup>1</sup> (Tables 1 and 2). The SDSI and RDSI increased at 10 mg L<sup>−</sup>1; in contrast, a negative correlation was observed with a further increase of the V level (25, 50, and 75 mg L<sup>−</sup>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<sup>−</sup>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 CO2 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−<sup>1</sup> 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 CO2 at a low level of V (10 mg L<sup>−</sup>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 CO2 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<sup>−</sup>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]. H2O2 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 H2O2 level (Figure 5B). Previous studies also observed a significant increment in the H2O2 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 H2O2 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<sup>−</sup>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 O2 •− 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]. Izbia ´nska 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.
