**1. 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−<sup>1</sup> 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−<sup>1</sup> [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

**Citation:** Kumar, S.; Wang, M.; Liu, Y.; Zhu, Z.; Fahad, S.; Qayyum, A.; Zhu, G. Vanadium Stress Alters Sweet Potato (*Ipomoea batatas* L.) Growth, ROS Accumulation, Antioxidant Defense System, Stomatal Traits, and Vanadium Uptake. *Antioxidants* **2022**, *11*, 2407. https://doi.org/10.3390/ antiox11122407

Academic Editor: Nafees A. Khan

Received: 25 October 2022 Accepted: 2 December 2022 Published: 5 December 2022

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**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

growth and development of plants [6,8]. V toxicity, bioaccumulation, and bioavailability rely on the oxidation state of vanadium. Vanadate is the toxic chemical compound of V, and at high concentrations hinders phenotypic, physiological, and biochemical processes of plants, eventually obstructing plant growth and yield [8–10].

V can be easily uptake by the plants from the soil; however, the effect is dependent on the V content in the soil. A low level of V treatment enhances plant growth, photosynthesis, and gas exchange elements [11,12]. Furthermore, Altaf et al. reported that 35 mg L−<sup>1</sup> V treatment caused a drastic decline in the biomass of rice plants [13]. Similarly, Chen et al. exposed *Ipomoea aquatica* to 0–2.50 mg L−<sup>1</sup> V treatments, and they concluded that concentration-dependent effects of V can be observed for the physiological properties and the plants may adapt to the toxicity of V [12]. Another study demonstrated that tobacco has good vanadium tolerance at < 2.0 mg L−<sup>1</sup> [10]. A high level of V treatment negatively influences plant growth and yield by increasing reactive oxygen species (ROS) production, hindering lipid membranes, antioxidant enzyme activity, the metabolic process, and gene expression [14,15]. The plants overcome oxidative stress and scavenge ROS by enhancing the production of osmolytes, antioxidants, and stimulating the activities of the antioxidant enzymes [16,17].

Previous studies highlighted the V effect on plant physiology by reducing or altering the photosynthetic rate, shoot and root survival, and leaf chlorosis [8,18]. Photosynthesis is a vital plant metabolic process that increases carbon absorption and yield production; however, its production and fixation can be strongly hindered under heavy metal stress [13,19]. Different studies reported a significant reduction in photosynthetic pigments and gas exchange elements in pepper and watermelon under heavy metals, including vanadium, nickel, and selenium stress [11,20]. Photosynthetic pigments can be impaired by obstructing electron transport and hindering the membrane integrity of the chloroplast. Similarly, plant roots are the essential organ that interacts with and absorbs metal and other components of the soil. Roots are considered the first line of defense and give structural support to the plant against heavy metal toxicity. It helps to defend itself by minimizing the absorption of unnecessary metal [21]. Previous studies also depicted that a higher level of V supplementation significantly reduced root architecture in several plants [11,13,22]. Different plants react differently to V stress, so more research is needed to determine the best concentration of V as a biostimulant in different cultivars of the crops.

Sweet potato (*Ipomoea batatas* L.) has been used as a major source of carbohydrate in many countries around the word, especially Asia and Africa [23]. Additionally, sweet potato roots are used for biofortification and bioethanol production. Thus, the production of sweet potato can be an integral part of food security in the future [24,25]. Moreover, sweet potato stems and leaves can be used as a vegetable for humans and animal feed [26,27]. In addition, sweet potato leaves are rich in protein, iron, calcium, fiber, carotenoids, vitamins, and total polyphenols and possess medicinal properties [28,29]. Sweet potato can severely affect productivity and quality under heavy metal stresses [23,30]. Several studies found that trace amounts of certain elements may stimulate the growth and production of horticultural crops [11,31]. Conversely, V application showed deleterious effects on the growth of different plant species by obstructing their antioxidant defense system [13,20]. However, the physiological and biochemical response of the sweet potato plant under V stress has not been well-studied and requires further research. Therefore, it is essential to explore the influence of V in sweet potato plants, primarily to determine the toxicity level of V for sweet potato. We designed this study to investigate V's effects on sweet potato plants and identify the toxic level of V. Our objective was to determine the effects of V on sweet potato growth, biomass, root morphology, photosynthesis, photosynthetic assimilation, antioxidant defense, stomatal traits, and V uptake.

#### **2. Methodology**

#### *2.1. 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−<sup>1</sup> 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 (25–27 ◦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 (NH4VO3). 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.

#### *2.2. 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:

$$\text{SDSI} = \frac{\text{Shoot DW (stressed plants)}}{\text{Shoot DW (controlled plants)}} \times 100\tag{1}$$

Similarly, the following formula was used to calculate the plant's RDSI;

$$\text{RDSI} = \frac{\text{Root DW (stressed plants)}}{\text{Root DW (controlled plants)}} \times 100\tag{2}$$

#### *2.3. Relative Water Content Analysis*

After recording the FW, the sweet potato leaves were immersed in ddH2O 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:

$$\text{RVC}^{\text{o}} \%= \left[ (\text{FW} - \text{DW}) / (\text{TW} - \text{DW}) \right] \times 100\tag{3}$$

#### *2.4. Root Morphology*

After surface rinsing, the roots were washed with ddH2O 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].

#### *2.5. 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].

#### *2.6. 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 HNO3, 0.5 mL H2O2, 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 <sup>μ</sup>g/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)

$$\text{Translation} = \frac{\text{V concentration in the plant shots}}{\text{V concentration in the plant roots}} \tag{5}$$

#### *2.7. 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].
