Titanium Nanoparticles (TiO₂-NPs) as Catalysts for Enhancing Drought Tolerance in Grapevine Saplings

Abstract

Drought is a major stress that hinders plant growth and causes water stress, posing a significant threat to global food security. While nanotechnology, particularly the use of nanoparticles such as TiO₂, offers a promising solution by enhancing plants’ resilience to drought stress, improving nutrient absorption, and promoting growth under adverse conditions, its application in viticulture remains underexplored. The objective of this research was to investigate the effects of titanium dioxide nanoparticles (TiO₂-NPs; 100, 10, 1, and 0 ppm (control)) on various physiological, biochemical, and morphological parameters in grapevine saplings. Three different rootstock varieties, 41 B/Crimson Seedless (CS), 1103 P/CS, and 5 BB/CS, were included in the experiment to assess how rootstock variety influences the response of grapevine saplings to TiO₂-NPs under drought stress (40–50%) and well-irrigated (90–100%) conditions. Young vines grown in pots under greenhouse conditions were used in this study. Applications of 10 ppm TiO₂-NPs improved growth parameters and the SPAD index and enhanced stomatal conductance, relative water content, and protein content in grapevine saplings under both drought and well-irrigated conditions. Conversely, oxidative stress parameters, including the membrane damage index, hydrogen peroxide, drought index, and lipid peroxidation levels, were significantly reduced following 10 ppm TiO₂-NP applications under drought conditions. Furthermore, total phenolic content, proline content, and ascorbate peroxidase, catalase, and superoxide dismutase activities, which increased significantly with drought stress, were reduced to lower levels, paralleling the alleviation of drought-induced oxidative stress. Our results suggest that the primary role of TiO₂ nanoparticles in enhancing drought tolerance is due to their beneficial effects in alleviating damage caused by drought stress. This finding applies not only to grapevines but may also be relevant for other agricultural crops.

1. Introduction

Climate change and global warming have led to increasingly frequent extreme weather events, including heat waves and drought, posing significant challenges to agriculture worldwide [1]. The viticulture sector, with its extensive production area of 7 million hectares and an annual output approaching 75 million tons across over 90 countries, is particularly vulnerable to these environmental stressors [2]. The IPCC (The Intergovernmental Panel on Climate Change) projects an increase in global average air and surface temperatures of 1–4.5 °C, which is expected to exacerbate drought conditions in many traditional viticultural regions, thereby increasing irrigation requirements [3]. Water scarcity, especially in semi-arid and arid climates, has become a key factor limiting vine productivity in agricultural systems [4]. Therefore, sustainable vineyard management requires a focus on improving water use efficiency rather than simply reducing water consumption [5]. The increasing vulnerability of water resources in many viticultural areas has sparked considerable interest among researchers in understanding grapevines’ responses to drought stress [6]. Grapevines have developed complex, interconnected mechanisms to cope with the adverse effects of drought, such as stomatal closure to reduce water loss, osmotic adjustment to maintain cell turgor, and changes in root architecture to enhance water uptake [7].

Drought stress profoundly affects various aspects of plant morphology, physiology, and biochemistry. Morphological effects include reduced shoot and root growth, decreased leaf number and size, and accelerated leaf senescence [8,9]. Physiologically, drought induces stomatal closure, leading to reduced transpiration, CO₂ conductance, chlorophyll content, and photosynthetic activity [10]. There are several studies that have claimed that TiO₂ NPs decreased the rate of photosynthesis, while the intercellular CO₂ level and stomatal conductance increased, suggesting a non-stomatal limitation of photosynthesis [11]. At the biochemical level, drought stress increases the production of reactive oxygen species (ROS), causing oxidative stress [12]. Plants respond by activating antioxidant defense mechanisms, including enzymatic antioxidants such as CAT and SOD, as well as non-enzymatic antioxidants such as phenolic compounds [13]. Several studies have demonstrated increased antioxidant enzyme activities in drought-stressed grapevines [14,15]. Polyphenolic compounds play a crucial role in the antioxidant defense system, supporting plants’ adaptation to stress conditions and mitigating ROS-induced damage [16]. However, prolonged and severe drought can inhibit total phenolic synthesis in grapevine leaves and roots [17], necessitating alternative approaches to enhance drought tolerance.

Nanotechnology, especially the use of nanoparticles (NPs), has emerged as a promising approach to tackle environmental challenges in agriculture due to their distinct physicochemical properties [18]. Recent papers have indicated that titanium dioxide nanoparticles (TiO₂-NPs) can enhance plants’ tolerance of various stress factors, including drought, by increasing antioxidant capacity [19,20]. Green synthesis of NPs offers not only an environmentally friendly but also a cost-effective alternative to traditional methods, utilizing plant-derived secondary metabolites as reducing and stabilizing agents [21,22]. Viticulture materials, such as grape seeds, stems, and skins, are rich sources of secondary metabolites and natural antioxidants, making them ideal candidates for the green synthesis of NPs [23,24]. Several studies have stated the potential of viticulture byproducts in the green synthesis of NPs, leveraging their polyphenolic compounds as reducing and stabilizing agents, as well as their antioxidant properties for ROS detoxification [25,26,27]. This approach not only offers a sustainable method for synthesizing NPs but also presents a potential solution for alleviating drought stress in grapevines and other agricultural crops. Considering these considerations, understanding the mechanisms of action of NPs is of paramount importance, particularly for grapevines, a crop with an extensive cultivation area and high economic value. Despite the increasing volume of research on NPs in agriculture, a comprehensive review of the existing studies highlighted a notable gap in evaluating the effects of TiO₂-NPs on defense mechanisms against drought stress in plants. The green synthesis of TiO₂-NPs, which utilizes eco-friendly methods involving natural materials such as plant extracts, not only minimizes the environmental impact but also enhances the nanoparticles’ biocompatibility and functional properties. This approach holds promise for developing sustainable solutions to improve plants’ resilience in the face of environmental stressors. This research gap presents a critical opportunity for investigation, given the increasing challenges posed by climate change to viticulture. The present study aimed to evaluate the effects of TiO₂-NPs, synthesized via a green synthesis method using vine leaves, as an elicitor of drought stress tolerance in grapevine saplings, and to detect the optimal concentration ranges of TiO₂-NPs for mitigating or potentially eliminating drought stress damage. In line with these aims, we hypothesized that TiO₂-NPs synthesized from vine leaves will significantly enhance drought stress tolerance in grapevine saplings compared with untreated controls, that there exists an optimal concentration range of TiO₂-NPs that maximizes drought stress mitigation without causing phytotoxicity, and that the application of TiO₂-NPs will lead to measurable improvements in specific physiological and biochemical markers associated with drought tolerance in grapevines. By exploring the intersection of nanotechnology and viticulture, this study contributes to the development of sustainable strategies for enhancing grapevines’ resilience to drought stress. Our findings aim to support the long-term viability of the viticulture sector in the face of changing climatic conditions, offering potential solutions to the challenges posed by increasing water scarcity in wine-growing regions.

2. Materials and Methods

2.1. Experimental Site and Plant Materials

This study was conducted between 2023 and 2024 in the research greenhouse and laboratories of the Faculty of Agriculture at Yozgat Bozok University. However, it is important to note that the stress treatments and NP applications, along with the analyses, were carried out within a single year. The synthesis and characterization of TiO₂-NPs, processes that involve the production of nanoparticles and the assessment of their physical, chemical, and structural properties, were conducted at the Science and Technology Application and Research Center (BILTEM) laboratories of Yozgat Bozok University, as well as at the Central Research Laboratory of Bartın University. The choice of table grape varieties for this research was based on their widespread cultivation under conditions of non-limiting water availability, which allowed for a clearer assessment of how drought stress impacts grapevines. Specifically, we utilized grapevine saplings of the Crimson Seedless (Vitis vinifera L.) cultivar, which is known for its economic importance and market appeal. The selected rootstocks were Kober 5 BB (V. berlandieri × V. riparia), known for its low drought tolerance; 41 B (41 B Millardet Et de Grasset) (Vitis vinifera L. cv. Chasselas × V. berlandieri), which exhibits moderate drought tolerance; and 1103 P (1103 Paulsen) (V. berlandieri × V. rupestris), recognized for its high drought tolerance.

2.2. Growing Grafted Grapevine Saplings

The scion and rootstock cuttings, retrieved from cold storage, were subjected to fungicide treatment by immersion in a fungicide solution for 48 and 24 h, respectively. After grafting, the rootstocks underwent bud elimination and base refreshment procedures, while the scions were trimmed to a single bud. The grafting process was executed using the Omega (Ω) system, with immediate application of the first paraffin layer to promote callus development at the graft union. Subsequently, the grafted cuttings were stratified in plastic crates, alternating layers of moist pine sawdust and paraffin-coated cuttings. The stratification crates were transferred to a climate-controlled chamber for a 21 d callusing period under conditions of 23 ± 2 °C in temperature and 85–90% relative humidity. After the callusing process was completed, the grafted cuttings were taken out of the crates, cleaned of sawdust with compressed air, and coated with paraffin again to protect the developed callus tissue from moisture loss. The cuttings were then moved to the greenhouse for planting.

2.3. Planting and Cultivation of Saplings

The experiment was conducted in a research greenhouse with an approximate area of 200 m², featuring a curved roof constructed of polycarbonate (PC) material. The facility was equipped with 55% shade cloth, a fan and pad system with fan heaters, and a ventilation system, all situated on a concrete foundation. The greenhouse contained rooting tables measuring 5 m in length, 80 cm in height, 1.20 m in width, and 20 cm in depth, designed to accommodate pots. Prior to planting, the grafted grapevine saplings underwent a quick-dip treatment with 2000 ppm indole butyric acid (IBA). They were then placed in polyethylene (PE) pots with dimensions of 11 × 11 × 22 cm (width × length × height), filled with a 1:1 mixture of sterile peat and perlite by volume. Immediately after planting, the growing medium was irrigated daily with a full-strength nutrient solution, following the recommendations of Ollat et al. [28] for the cultivation of grapevine saplings. The solution’s composition included Ca(NO₃)₂.4H₂O (2.5 mM), KH₂PO₄ (1.0 mM), KNO₃ (2.5 mM), MgSO₄.7H₂O (1.0 mM), ZnSO₄.7H₂O (2.40 µM), Na₂MoO₄ (0.013 µM), CuSO₄ (0.5 µM), MnCl₂.4H₂O (9.2 µM), NaFe(III)-EDTA (45 µM), and H₃BO₃ (46.4 µM), with the pH adjusted to 6.5. The irrigation volume was calculated based on a 30% drainage ratio and maintained until the onset of the drought stress treatments, which lasted for a duration of three months. During the vegetation period, the greenhouse environment was controlled at a temperature of 25 ± 5 °C, with a relative humidity of 40 ± 10% and natural day length conditions. A heater, along with a fan and pad system, was used to optimize root and shoot development in the grafted saplings. A single shoot was allowed to develop on each grafted grapevine sapling prepared from single-bud cuttings. Approximately eight weeks post-planting, upon achieving sufficient root and shoot development, saplings exhibiting similar stem diameter and leaf area characteristics were selected. The plants were then transplanted into 2.5 L polypropylene (PP) pots with dimensions of 12.5 × 20 × 12.5 cm, filled with a soil, perlite, and peat mixture in a ratio of 1:1:1.

2.4. Characterization of TiO₂-NPs and Green Synthesis

Grapevine leaves were utilized as reducing agents in the green synthesis of TiO₂-NPs. The leaves were collected in June, during the early growth stages, with the fifth leaf from the shoot tip being specifically selected. The collected leaves were subjected to drying in a shaded environment to standardize the moisture levels. To prepare the leaf extracts, the dried grapevine leaves were first rinsed with distilled water, dried again, and then ground into small fragments using liquid nitrogen. A 30 g sample of the fragmented leaves was extracted in 100 mL of distilled water at 60 °C for 1 h using a magnetic stirrer. Once cooled to room temperature, the mixture was centrifuged at 3500× g rpm for 15 min and then filtered through Whatman No. 1 filter paper [29]. The resulting leaf extracts were stored at 4 °C for use in TiO₂-NP synthesis. Commercial titanium tetrachloride (TiCl₄) (CAS RN: 7550-45-0) from TCI was employed as the TiO₂ source. The synthesis of TiO₂-NPs involved treating 50 mL of a 4 mM titanium tetrachloride solution with 2 mL of the leaf extract, followed by heating at 80 °C for 24 h under magnetic stirring. The precipitate solution was then centrifuged at 10,000× g rpm for 15 min and washed twice with deionized water. After overnight drying in a 60 °C oven and calcination at 500 °C for 5 h, the nanoparticles were characterized [30]. The synthesized nanoparticles were characterized using UV–vis spectrophotometry, X-ray diffraction (XRD; Malvern Panalytical, Empyrean, Malvern, UK), scanning electron microscopy (SEM; FEI Quanta FEG 450, Hillsboro, OR, USA), and energy-dispersive X-ray spectroscopy (EDX; FEI Quanta FEG 450, Hillsboro, OR, USA).

2.4.1. UV–Vis Analysis

Ultraviolet–visible spectroscopy was employed to measure the molecules, inorganic ions, and complexes present in the solution samples extracted during the green synthesis reaction.

2.4.2. XRD Analysis of Powder

X-ray diffraction analysis was performed on powder samples to elucidate the crystalline structure of the synthesized nanoparticles. Additionally, this technique facilitated the determination of essential parameters for calculating the average particle size, offering crucial information about the physical properties of the TiO₂ nanoparticles.

2.4.3. SEM Analysis

Scanning electron microscopy was utilized to obtain high-resolution, high-magnification images of the powder samples. This imaging technique enabled the visualization of the TiO₂ nanoparticles’ surface morphology, providing detailed information about the number, size, and shape of the particles.

2.4.4. EDX Analysis

Energy-dispersive X-ray spectroscopy was conducted to perform a quantitative chemical analysis by leveraging the characteristic energies of elements. In this study, EDX analysis was specifically employed to gather information about the elemental composition and purity of the TiO₂ samples, offering a comprehensive understanding of the synthesized nanoparticles’ chemical nature.

2.5. Preparation and Application of NP Solutions

In accordance with a previous study [31] that demonstrated significant enhancements in stress-induced physiological processes when NPs were applied prior to stress exposure, TiO₂-NPs were administered before the onset of drought stress in this research. The TiO₂-NPs obtained through green synthesis were considered to be pure, and stock solutions were initially prepared at 1000 ppm concentration. These stock solutions were subsequently diluted to 1, 10, and 100 ppm concentrations. Distilited water was used for the control groups. After transplantation, solutions of TiO₂-NPs at concentrations of 0, 1, 10, and 100 ppm were uniformly applied to the entire green surface of the grapevine saplings via a foliar spray, with 25 mL of the solution per plant [32]. According to literature indicating that repeated applications of elicitors are more effective when administered at regular intervals [31,33], TiO₂-NPs were applied three times at one-week intervals in this study.

2.6. Drought Stress Application

About four weeks after the TiO₂-NP treatments, grapevine saplings were exposed to drought stress using the approach described by Cochetel et al. [34]. The irrigation schedule consisted of two treatments: a control group (maintained at 90–100% field capacity) and a drought stress group (40–50% field capacity) [33,35]. To calculate daily water loss, the gravimetric substrate water content (GSWC) method was applied to a representative sample of the growing medium, using the formula provided by Earl [36]:

GSWC (%) = (substrate wet weight − substrate dry weight)/(substrate dry weight) ×100

(1)

To ensure that the growing media maintained the targeted moisture content throughout the experiment, the pot weight and moisture levels were monitored daily during the 30-day drought stress application period. Water supplementation was performed daily at the specified rates until the conclusion of the experiment. Additionally, all plants received a daily application of the full-strength nutrient solution [28] throughout the trial period. The growing medium used in the experiment was prepared at a medium density and not overly compacted to ensure the proper air balance for plant roots. To determine 100% field capacity, a homogeneous mixture of 2.5 L of a soil:peat:perlite medium was fully saturated with water and allowed to drain. Upon completion of drainage, the substrate at 100% field capacity was weighed, then oven-dried at 105 °C and reweighed. Using the GSWC (%) formula presented above, the water volume required to achieve 100% field capacity for the growing medium was calculated as 1.5 L. Before the commencement of the drought stress treatments, all plants were irrigated daily with approximately 120 mL of water to compensate for the daily water consumption of the plants and evaporative losses, which were estimated to be at around 8%. Upon commencement of the drought stress treatments, the daily water volume provided to the plants was 115 mL to maintain 90–100% field capacity and 55 mL to maintain 40–50% field capacity. These volumes increased towards the end of the experiment due to the plants’ growth and the corresponding increased water consumption, reaching 125 mL for 90–100% field capacity and 60 mL for 40–50% field capacity. In all irrigation applications, the volume of the 20 mL nutrient solution provided daily for fertilization was subtracted from the calculated total water volume.

2.7. Measurements and Analyses in Saplings

The trial was concluded after a 120-day cultivation period, which spanned from May to August. Some morphological, physiological, and biochemical parameters were analyzed to detect differences among the treatments. Parameters such as shoot length, leaf count, physical damage, stomatal conductance, chlorophyll content, and leaf surface temperature were examined immediately prior to the experiment’s conclusion. Physical damage was assessed by evaluating the visual symptoms of drought stress on the grapevine saplings, including leaf wilting, discoloration, and overall plant vigor. Shoot fresh and dry weights, leaf area, root length, shoot dry matter ratio, root fresh and dry weights, root dry matter ratio, leaf relative water content, and the membrane damage index were measured immediately after the plants were removed from their growth media at the experiment’s end. The fourth leaf from the top of the shoots was chosen for determining the leaf area and conducting physiological and biochemical measurements because it typically represents a consistent and mature stage of leaf development, minimizing the variability associated with younger or older leaves. Samples for biochemical analyses, including hydrogen peroxide content, total phenolic content, proline content, soluble protein content, lipid peroxidation, and antioxidant enzyme activities (CAT, APX, and SOD), were collected immediately after the conclusion of the experiment. These samples were subsequently stored in an ultra-deep freezer at −80 °C (Forma-89000 Series, Thermo Scientific, Waltham, MA, USA).

2.7.1. Morphological Analyses

The leaf count was determined by enumerating all leaves from the first fully opened leaf at the shoot tip, designated as the first leaf, downwards to the base. Leaf area (cm²) was measured utilizing a leaf area meter (ADC BioScientific Area Meter AM-300). Shoot length (cm) was determined by measuring the distance from the shoot tip to the base using a ruler. Root length (cm) was assessed by measuring the distance between the emergence point of the longest root and its terminus using a ruler. Fresh weights (g) of shoots and roots were determined using an analytical balance (AXIS ACN220, Axis, Gdańsk, Poland) with 0.0001 g precision. Roots and shoots were kept in an air-circulating oven at 65 °C for 72 h after determining the fresh weights, then weighed using an analytical balance to determine the dry weights (g). The dry matter ratios of shoots and roots were calculated as a percentage by dividing the dry weights by the fresh weights. The evaluation of chlorosis damage in leaves was conducted using a 1–5 scale to provide a standardized and quantifiable assessment of the severity of leaf discoloration [37]: 5—very severe chlorosis (yellow leaves with more than 10% necrosis); 4—severe chlorosis (yellow leaves with less than 10% necrosis); 3—moderate chlorosis (main veins green, areas between veins yellow); 2—mild chlorosis (light green between veins); 1—no chlorosis (dark green leaves). The degree of physical damage in plants was calculated as a drought index percentage using the aforementioned 1–5 scale according to the following formula. The percentage values for well-irrigated controls showing no drought symptoms were subtracted from the results of all treatments under drought stress.

Drought index (%) = Σ(leaf × scale value)/(total leaves × highest scale value) × 100

(2)

2.7.2. Physiological Analyses

Chlorophyll content was measured using a portable chlorophyll meter (Konica Minolta SPAD-502, Konica Minolta Optics, Japan), focusing on two areas near the main vein of the leaves. Stomatal conductance (mmol m⁻² s⁻¹) and leaf surface temperature (°C) were assessed by placing the sensor of a porometer (SC⁻¹ Leaf Porometer, Decagon, Pullman, WA) on the underside of the leaf between the veins. This method ensured accurate measurements of the leaf’s physiological parameters. Leaf relative water content (RWC%) was calculated using the following formula, utilizing the fresh weight (FW), turgid weight (TW) after 6 h in distilled water, and dry weight (DW) after 24 h in an air-circulating oven at 80 °C [38,39]:

RWC (%) = [(FW − DW)/(TW − DW)] × 100

(3)

To assess the membrane damage index, three leaf discs, each 6 mm in diameter, were cut using a cork borer and immersed in 20 mL of deionized water for 4 h. The initial electrical conductivity (EC₁) was measured using an EC meter (Jenway 470 conductimeter, Jenway 4071, UK). Afterward, the same discs were incubated at 100 °C for 10 min, and the final electrical conductivity (EC₂) was recorded. Electrolyte leakage (EL) was then calculated using the following formula [40]:

EL (%) = (Lt − Lc/1 − Lc) × 100

(4)

where Lt is the EC₁/EC₂ value of drought-stressed leaf, and Lc is the EC₁/EC₂ value of control leaf.

2.7.3. Biochemical Analyses

The proline content was determined following the method of Bates et al. [41]. Leaf samples were homogenized in sulfosalicylic acid and centrifuged, and the supernatant was mixed with glacial acetic acid and acidic ninhydrin before incubation and absorbance measurements at 520 nm. For total phenolic content, the extraction and analysis were performed according to Kiselev et al. [42] and Singleton and Rossi [43], using ethanol extraction and the Folin–Ciocalteu method, with the results expressed as gallic acid equivalents. Hydrogen peroxide content was measured as per Velikova et al. [44], using cold TCA (trichloroacetic acid) extraction and spectrophotometric analysis at 390 nm. Lipid peroxidation, quantified as malondialdehyde, followed the method of Lutts et al. [45], using TCA extraction, incubation with TBA (thiobarbituric acid), and absorbance readings at 532 and 600 nm. Protein content and antioxidant enzyme activities were measured using extracts prepared in a phosphate buffer with EDTA and PVP, following Özden et al. [46]. The stability of APX was ensured by adding ascorbate to the buffer. Soluble protein content was determined using Bradford’s method [47], with absorbance at 595 nm. Superoxide dismutase (SOD) activity was assessed based on Agarwal and Pandey’s method [48], involving NBT reduction, while catalase (CAT) activity was determined according to Gong et al. [49], monitoring the decrease in H₂O₂ absorbance at 240 nm. Ascorbate peroxidase (APX) activity was measured following Nakano and Asada [50], based on the oxidation of ascorbate and absorbance monitoring at 290 nm.

2.8. Experimental Design and Data Analysis

All experiments were carried out using a three-factor completely randomized design (CRD). The experiment was structured as a factorial trial with three factors, with the rootstock/scion combination (R/S) as the first factor, the irrigation regime as the second factor, and the NP concentration as the third factor. The treatments compared in this study included three rootstock/scion combinations, namely 5 BB (low drought tolerance), 41 B (moderate drought tolerance), and 1103 P (high drought tolerance), all grafted with Crimson Seedless (Vitis vinifera L.) as the scion. The two irrigation regimes were the control (90–100% field capacity) and drought stress (40–50% field capacity). The four NP concentrations were 0 ppm (control), 1 ppm, 10 ppm, and 100 ppm of TiO₂-NPs. This resulted in a total of 24 treatment combinations (3 × 2 × 4). Each parameter was evaluated with three technical replicates, involving nine plants per replicate. Numerical data were recorded using Microsoft 365 Excel (Office 2021 and Microsoft 365). A three-way analysis of variance (three-way ANOVA) was performed using IBM SPSS version 22.0 software to assess the interactions among the three independent variables. Post hoc comparisons were conducted with Duncan’s multiple range test to determine the mean separation between treatments at the 5% significance level (p ≤ 0.05). Data are presented as the mean values with the corresponding standard deviations (SD). To better elucidate the relationships between the treatments and the examined traits, correlation analysis was performed using the SRPLOT online platform (https://www.bioinformatics.com.cn/en, accessed on 16 August 2024). Additionally, a hierarchical clustering heatmap was generated to visualize the relationships and intensities between the factors and the examined traits. Furthermore, principal component analysis (PCA) was conducted using GraphPad Prism version 9.3.1 (GraphPad Software, LLC, San Diego, CA, USA) to determine the direction of the relationships between the factors and the examined traits, with the results explained using a biplot following the methodology outlined by Evgenidis et al. [51].

3. Results

3.1. Characterization Analysis of TiO₂-NPs

In our study, the characterization analysis of TiO₂-NPs biosynthesized using grapevine leaf extracts yielded significant findings. UV–vis analysis revealed a maximum absorption band at approximately 318 nm (Figure 1A). X-ray diffraction (XRD) analysis was employed to investigate the structure, crystalline purity, and average size of the TiO₂-NPs. The XRD pattern (Figure 1B) indicated that the TiO₂-NPs synthesized using grapevine as a reducing agent exhibited an anatase crystal structure. Anatase, a metastable mineral form of TiO₂, is characterized by a tetragonal crystal structure. Examination of the XRD patterns revealed diffraction angles at 2θ values of 25.29°, 37.86°, 47.9°, 54.01°, 55.12°, 62.54°, 68.64°, and 70.08°, corresponding to the 011, 004, 020, 015, 121, 024, and 220 Bragg reflection planes, respectively. The average crystal size of the synthesized nanoparticles was calculated to be 37 nm using the Scherrer equation (D = Kλ/βcosθ). Scanning electron microscopy (SEM) analysis was conducted to examine the surface morphology and size of the TiO₂-NPs. The results revealed that the TiO₂-NPs were spherical in shape and exhibited a heterogeneous distribution ranging from 16 to 23 nm (Figure 1C). These heterogeneous structures were observed to form layered aggregates, which presented challenges in examination of the nanoparticles. The particle size measurements obtained from SEM were in agreement with the average crystal size measured by XRD analysis. To further analyze the sample, Energy-dispersive X-ray spectroscopy (EDX) was conducted to identify the elemental composition of the chemical compounds present (Figure 1D). The synthesized sample primarily consisted of titanium (66.3%) and oxygen (31.8%). It was concluded that the titanium content in the plant-mediated TiO₂-NPs was higher compared with the oxygen content. Additionally, trace amounts of chlorine (Cl) and potassium (K) were detected, which were presumed to be of plant origin.

3.2. Defense Mechanisms in Grapevine Saplings

The three-way ANOVA results revealed the significant effects of the main factors of the rootstock/scion combination (R/S) (R/S combinations, irrigation regime, and TiO₂-NP concentrations) and their interactions on same biochemical, morphological, and physiological characteristics of grapevine saplings (Table S1). The analysis of variance revealed significant interactions (p ≤ 0.05) among the factors of rootstock/scion (R/S) combination, irrigation regime, and TiO₂-NP concentration for several traits. These traits included leaf relative water content, root fresh weight, shoot dry weight, root dry weight, stomatal conductance, drought index, electrolyte leakage, proline content, H₂O₂ production, protein amount, total phenolic content, and antioxidant enzymes (SOD, CAT, and APX). For root length, significant interactions were found for “R/S combination × irrigation regime”, “R/S combination × TiO₂-NP concentration”, and “irrigation regime × TiO₂-NP concentration”. In terms of shoot fresh weight, root leaf area, dry matter ratio, and the SPAD index, significant interactions occurred for “R/S combination × irrigation regime” and “irrigation regime × TiO₂-NP concentration”. Leaf temperature was significantly affected by the interactions “R/S combination × TiO₂-NP concentration” and “irrigation regime × TiO₂-NP concentration”. The interaction of “irrigation regime × TiO₂-NP concentration” was significant for leaf number, shoot dry matter ratio, and malondialdehyde content. Shoot length was significantly influenced by the factors R/S combination, irrigation regime, and TiO₂-NP concentration. The traits affected by all three factors included leaf area, shoot length, shoot fresh weight, root length, the SPAD index, and leaf temperature. The root dry matter ratio was significantly influenced by the R/S combination and NP concentration, while leaf number and malondialdehyde content were significantly affected by the irrigation regime and NP concentration. Lastly, the irrigation regime significantly impacted the shoot dry matter ratio.

3.2.1. Impact of TiO₂-NPs on Growth Traits

Drought stress significantly reduced shoot length compared with well-irrigated conditions (17.92 cm vs. 29.66 cm, p < 0.001). The 10 ppm TiO₂-NP treatment yielded the highest shoot length (25.89 cm), while the control and 100 ppm treatments resulted in the lowest values (22.85 and 22.41 cm, respectively). Among the R/S combinations, 41 B/CS and 1103 P/CS exhibited the highest shoot lengths (23.85 and 24.37 cm, respectively) (Figure 2A, Table S11). Shoot fresh weight significantly decreased under drought stress (6.38 g vs. 9.17 g in well-irrigated conditions, p < 0.001). The 10 ppm TiO₂-NP treatment produced the highest shoot fresh weight (8.80 g), while the control and 1 and 100 ppm treatments resulted in lower values (7.29, 7.66, and 7.34 g, respectively). The 1103 P/CS combination showed the highest shoot fresh weight (8.30 g) among the R/S combinations (Figure 2B, Table S8).

Under drought conditions, shoot dry weight significantly decreased for all R/S combinations (p = 0.025). The highest value was observed in well-irrigated 1103 P/CS with 10 ppm TiO₂-NP (4.04 g), while the lowest values were found in the drought-stressed control treatments for all combinations (5 BB/CS, 1.59 g; 41 B/CS, 1.67 g; 1103 P/CS, 1.66 g) (Figure 2C, Table S2). Drought stress significantly reduced the shoot dry matter ratio (28.19% vs. 32.97% in well-irrigated conditions, p < 0.001). The highest values were observed in well-irrigated conditions with the control, 10 ppm, and 100 ppm TiO₂-NP treatments (32.81%, 35.04%, and 32.91%, respectively) (Figure 2D, Table S8). Leaf number decreased significantly under drought stress (7.48 vs. 9.56 in well-irrigated conditions, p < 0.001). The 10 ppm TiO₂-NP treatment resulted in the highest leaf number (9.34), while the control and 1 ppm treatments showed the lowest values (8.31 and 8.50, respectively) (Figure 2E, Table S7). Leaf area was significantly reduced by drought stress (51.44 cm² vs. 68.53 cm² in well-irrigated conditions, p < 0.001). The 10 ppm TiO₂-NP treatment yielded the highest leaf area (63.07 cm²), while the control and 1 ppm treatments resulted in the lowest values (59.25 and 60.50 cm², respectively) (Figure 2F, Table S7).

Root length decreased significantly under drought stress (23.01 cm vs. 25.79 cm in well-irrigated conditions, p < 0.001). The 10 ppm TiO₂-NP treatment produced the longest roots (27.41 cm), while the control and 1 ppm treatments resulted in the shortest roots (23.61 and 24.19 cm, respectively). The 1103 P/CS combination exhibited the longest roots (25.53 cm) among the R/S combinations (Figure 3A, Table S9). Root fresh and dry weights were significantly reduced by drought stress in all R/S combinations (p < 0.001 and p = 0.001, respectively). The highest root fresh and dry weights were observed in well-irrigated conditions with the 10 ppm TiO₂-NP treatment for all combinations (Figure 3B,C, Table S2). The root dry matter ratio was not significantly affected by the irrigation regime (p = 0.090). However, TiO₂-NP treatments (1, 10, and 100 ppm) resulted in higher root dry matter ratios (60.56%, 62.61%, and 59.73%, respectively) compared with the control (56.88%). The 5 BB/CS combination showed the highest root dry matter ratio (65.63%) among the R/S combinations (Figure 3D, Table S9).

3.2.2. Impact of TiO2-NPs on Physiological Parameters

Compared with well-irrigated conditions, relative water content (RWC) decreased significantly across all rootstock/scion combinations under drought stress (p < 0.001). The highest RWC values were recorded for 1103 P/CS under well-irrigated conditions with the 10 ppm TiO₂-NP application (76.71%), while the lowest values were observed for 41 B/CS and 5 BB/CS under drought conditions with the 100 ppm TiO₂-NP treatment (37.85% and 39.05%, respectively) (Figure 3E, Table S3). Stomatal conductance significantly decreased under drought stress for all rootstock/variety combinations compared with well-irrigated conditions (p < 0.001). The highest values were observed for 1103 P/CS under well-irrigated conditions with the 10 ppm TiO₂-NP treatment (113.64 mmol m⁻² s⁻¹), whereas the lowest values were recorded for 5 BB/CS and 41 B/CS under the same conditions with the 100 ppm TiO₂-NP treatment (72.24 mmol m⁻² s⁻¹ and 74.16 mmol m⁻² s⁻¹, respectively) (Figure 3F, Table S3). The SPAD index values decreased significantly under drought stress (31.98 SPAD) compared with well-irrigated conditions (36.03 SPAD, p < 0.001). The 10 ppm TiO₂-NP treatment resulted in the highest SPAD values (37.75 SPAD), while the control group exhibited the lowest average (29.77 SPAD). Among the rootstock/variety combinations, 5 BB/CS had the highest average (35.07 SPAD). In terms of irrigation regime and rootstock/variety interactions, the highest SPAD values were found for 1103 P/CS and 5 BB/CS under well-irrigated conditions (36.68 and 36.50 SPAD, respectively), while the lowest averages were observed in 1103 P/CS and 41 B/CS under drought conditions (31.59 and 30.88 SPAD, respectively). Regarding the NP concentration and irrigation regime interactions, the highest SPAD values were recorded for 10 ppm TiO₂-NP under well-irrigated conditions (39.25 SPAD), while the lowest averages were found for 100 ppm TiO₂-NP under drought conditions (26.59 SPAD) (Figure 4A, Table S10). Leaf temperature increased significantly under drought stress compared with well-irrigated conditions (27.14 °C versus 25.60 °C, p < 0.001). The highest temperature was observed under the 100 ppm TiO₂-NP treatment (26.99 °C), while the lowest was recorded in the control group (25.97 °C). Among the rootstock/variety combinations, the lowest averages were for 1103 P/CS and 41 B/CS (26.25 and 26.34 °C, respectively). Among the rootstock/variety and NP concentration interactions, the highest value was found for 5 BB/CS with 100 ppm TiO₂-NP (27.31 °C), and the lowest for 1103 P/CS in the control group (25.83 °C). For the interaction between irrigation regime and NP concentration, the lowest temperature was observed in the well-irrigated control group (24.80 °C), while the highest temperature was recorded with 100 ppm TiO₂-NP under drought conditions (27.86 °C) (Figure 4B, Table S10). Drought stress caused a significant increase in the drought index across all rootstock/variety combinations compared with well-irrigated conditions (p < 0.001). The highest value was recorded for 5 BB/CS under drought conditions in the control group (56.78%), while the lowest values for all rootstocks were recorded in all treatments under well-irrigated conditions (0.00%) (Figure 4C, Table S3). Electrolyte leakage (EL) values increased under drought stress across all rootstock/variety combinations (p = 0.014). The lowest values were found in the control group for all rootstocks under well-irrigated conditions (41 B/CS, 5.34%; 1103 P/CS, 5.16%; 5 BB/CS, 5.36%), under the 1 ppm TiO₂-NP treatment (41 B/CS, 5.36%; 5 BB/CS, 5.48%; 1103 P/CS, 6.15%), and in 5 BB/CS with 10 ppm TiO₂-NP (6.42%). The highest values were recorded for all rootstocks under the 100 ppm TiO₂-NP treatment under drought conditions (5 BB/CS, 28.37%; 41 B/CS, 27.74%; 1103 P/CS, 28.70%). The most effective treatment in reducing EL under-drought conditions for all rootstocks was 10 ppm TiO₂-NP (5 BB/CS, 16.69%; 41 B/CS, 16.57%; 1103 P/CS, 16.38%) (Figure 4D, Table S4).

3.2.3. Effects of TiO₂-NPs on Biochemical Parameters

Compared with well-irrigated conditions, H₂O₂ levels significantly increased in all three R/S combinations under drought stress (p = 0.002). The highest H₂O₂ content was observed in the 100 ppm TiO₂-NP applications under drought conditions for all combinations (5 BB/CS, 32.35 µmol g⁻¹; 41 B/CS, 32.07 µmol g⁻¹; 1103 P/CS, 33.44 µmol g⁻¹). The lowest values were obtained under the control, 1 ppm, and 10 ppm TiO₂-NP treatments under well-irrigated conditions across all combinations (Figure 4E, Table S4). Moreover, MDA levels were significantly higher under drought stress compared with well-irrigated conditions, with values of 2.58 nmol g⁻¹ and 7.86 nmol g⁻¹, respectively (p < 0.001). Among the different concentrations, the highest MDA content was found under the 100 ppm TiO₂-NP treatment (7.73 nmol g⁻¹), while the lowest was observed under the 1 ppm and 10 ppm TiO₂-NP treatments (4.21 nmol g⁻¹). The lowest MDA content overall was under the control treatment under well-irrigated conditions (1.51 nmol g⁻¹), and the highest was under the 100 ppm TiO₂-NP treatment under drought conditions (11.17 nmol g⁻¹) (Figure 4F, Table S11). Drought stress resulted in significant increases in proline levels for all three R/S combinations when compared with well-irrigated conditions (p < 0.001). The highest proline content was observed under the 100 ppm TiO₂-NP treatments under drought conditions for 5 BB/CS and 41 B/CS (2.85 and 2.91 µmol g⁻¹, respectively). The lowest values were recorded under the control treatments under well-irrigated conditions (5 BB/CS, 0.35 µmol g⁻¹; 41 B/CS, 0.39 µmol g⁻¹; 1103 P/CS, 0.35 µmol g⁻¹) and under the 1 ppm TiO₂-NP treatment for 5 BB/CS under well-irrigated conditions (0.46 µmol g⁻¹) (Figure 5A, Table S4). Drought stress led to significant increases in total phenolic content across all R/S combinations compared with well-irrigated conditions (p = 0.028). The highest values were obtained for the 100 ppm TiO₂-NP treatments under drought conditions for all combinations (41 B/CS, 22.82 mg g⁻¹; 5 BB/CS, 22.85 mg g⁻¹; 1103 P/CS, 22.93 mg g⁻¹). The lowest phenolic content was found under the control treatments under well-irrigated conditions for all combinations (1103 P/CS, 4.05 mg g⁻¹; 5 BB/CS, 3.21 mg g⁻¹; 41 B/CS, 3.63 mg g⁻¹) (Figure 5B, Table S5). Compared with well-irrigated conditions, drought stress significantly reduced the soluble protein content in all R/S combinations (p < 0.001). The highest protein content was recorded under the 10 ppm TiO₂-NP treatment under well-irrigated conditions for 1103 P/CS (4.13 mg). The lowest protein levels were found under the control treatments under drought conditions for all combinations (5 BB/CS, 0.56 mg; 41 B/CS, 0.57 mg; 1103 P/CS, 0.64 mg), as well as under the 100 ppm TiO₂-NP treatments (5 BB/CS, 0.62 mg; 41 B/CS, 0.67 mg; 1103 P/CS, 0.50 mg) and under the 1 ppm TiO₂-NP treatment for 41 B/CS (0.65 mg) (Figure 5C, Table S5). Drought stress led to significant increases in SOD, CAT, and APX activity in all R/S combinations compared with well-irrigated conditions (p < 0.001). The highest SOD activity was observed under the control treatment under drought conditions for 5 BB/CS (81.10 U mg⁻¹ protein). The lowest values were found under the control treatments under well-irrigated conditions across all combinations (5 BB/CS, 21.96 U mg⁻¹ protein; 41 B/CS, 21.34 U mg⁻¹ protein; 1103 P/CS, 20.63 U mg⁻¹ protein) (Figure 5D, Table S5). The highest CAT activity was observed under the 100 ppm TiO₂-NP treatments under drought conditions for both 41 B/CS and 1103 P/CS, with a value of 0.48 U mg⁻¹ protein. The lowest values were recorded under the control treatments under well-irrigated conditions across all combinations (0.02 U mg⁻¹ protein) and under the 1 ppm TiO₂-NP treatments under the same conditions (0.04 U mg⁻¹ protein) (Figure 5E, Table S6). The highest APX activity was observed under the 100 ppm TiO₂-NP treatment under drought conditions for 1103 P/CS (26.57 U mg⁻¹ protein). The lowest values were recorded under all treatments under well-irrigated conditions (0.21–0.93 U mg⁻¹ protein) (Figure 5F, Table S6).

3.3. General Evolution

Pearson correlation analysis revealed strong positive correlations among most plant growth parameters, except for the root dry matter ratio (Figure 6). The highest positive correlations were noted between shoot fresh weight and shoot dry weight (0.98), between shoot length and leaf area (0.98), and between shoot dry weight and root dry weight (0.97). On the other hand, the strongest negative correlations were found between shoot length and drought index (−0.98), between leaf area and drought index (−0.96), and between leaf number and drought index (−0.90). Among the physiological parameters, the strongest positive correlations were observed between electrolyte leakage and leaf temperature (0.93), between stomatal conductance and the SPAD index (0.87), and between RWC and stomatal conductance (0.79). The strongest negative correlations were between electrolyte leakage and RWC (−0.93), between leaf temperature and RWC (−0.87), and between electrolyte leakage and the SPAD index (−0.79). For the biochemical parameters, the highest positive correlations were between hydrogen peroxide content and total phenolic content (0.97), between total phenolic content and MDA content (0.97), and between MDA content and H₂O₂ content (0.95). On the other hand, the strongest negative correlations were identified between hydrogen peroxide content and soluble protein content (−0.91), between total phenolic content and soluble protein content (−0.89), and between soluble protein content and MDA content (−0.87). The strongest positive correlations between plant growth and physiological parameters were observed between shoot length and RWC (0.97), between leaf number and RWC (0.97), and between RWC and leaf area (0.96). The most significant negative correlations were between shoot length and electrolyte leakage (−0.94), between electrolyte leakage and leaf area (−0.93), and between RWC and drought index (−0.93). In terms of correlations between sapling growth and biochemical parameters, the strongest positive relationships were found between shoot length and soluble protein content (0.98), between soluble protein content and leaf area (0.96), and between soluble protein content and root dry weight (0.94). The strongest negative correlations were observed between hydrogen peroxide content and shoot length (−0.95), between hydrogen peroxide content and leaf area (−0.95), and between soluble protein content and drought index (−0.95). Among the physiological and biochemical properties, the strongest positive correlations were identified between hydrogen peroxide content and electrolyte leakage (0.98), between MDA content and electrolyte leakage (0.98), and between total phenolic content and electrolyte leakage (0.97). Conversely, the most significant negative correlations were determined between hydrogen peroxide content and RWC (−0.94), between total phenolic content and RWC (−0.92), and between MDA content and RWC (−0.91).

To comprehensively evaluate the agronomic, physiological, and biochemical parameters, the experimental data were subjected to PCA. The first two principal components with eigenvalues > 1.0 explained 85.49% of the total variance. The high variance ratio explained by these components suggests that the evaluated variables are explained well by the component analysis. Using the scores from the first two components, the experimental groups were plotted in a biplot (Figure 7). All well-irrigated control groups, with the exception of 5BB/CS-WI-100, were positioned in the negative region of the first principal component (PC1), which accounted for 78.46% of the total variation. These groups generally exhibited higher averages in the growth parameters (excluding the root dry matter ratio), soluble protein content, and specific physiological traits such as relative water content (RWC), stomatal conductance, and the SPAD index. Among the well-irrigated applications, the 10 ppm TiO₂-NP treatment of 1103 P/CS exhibited the highest performance compared with the other treatments, followed by the 10 ppm TiO₂-NP treatments of 41 B/CS and 5 BB/CS under the same conditions. In contrast, all drought control groups (except 5BB/CS-DS-10 and 1103P/CS-DS-10) were positioned in the positive loading of PC1. These groups were characterized by higher averages in the oxidative stress parameters (MDA and H₂O₂ content, electrolyte leakage, drought index, and leaf temperature), proline and total phenolic content, and antioxidant enzyme activities (SOD, CAT, and APX). The control treatments without a TiO₂-NP application and the 100 ppm TiO₂-NP treatments in all R/S combinations under drought stress stood out among these groups. The second principal component (PC2), explaining 8.7% of the total variation, showed that the 10 ppm TiO₂-NP treatments of 5BB/CS and 1103 P/CS under drought conditions outperformed drought controls, with significantly lower oxidative stress values.

To visualize and clarify the findings and to relate them to the experimental groups, a hierarchical clustering heatmap was generated (Figure 8). The clustering of the heatmap separated the evaluated traits into two main clusters. The first main cluster grouped plant growth parameters, soluble protein content, RWC, stomatal conductance, and the SPAD index, while the second cluster included drought index, electrolyte leakage, antioxidant enzyme activities, hydrogen peroxide content, leaf temperature, root dry matter ratio, MDA, proline, and total phenolic content. The heatmap generally separated the applications into two main clusters: well-irrigated and drought-stressed conditions. The first cluster included drought treatments represented by lower averages in plant growth parameters, soluble protein content, and RWC, but higher averages in stress parameters such as antioxidant enzyme activities, drought index, electrolyte leakage, leaf temperature, hydrogen peroxide content, MDA, proline, and total phenolic content. Within the first main cluster, the 10 ppm TiO₂-NP treatments of all three R/S combinations under drought conditions formed a distinct subgroup characterized by notably low oxidative stress values and significantly higher growth performance. In the second subgroup of the first cluster, the 1 ppm TiO₂-NP treatment of 5BB/CS under drought conditions formed a separate branch, while the 100 ppm TiO₂-NP treatments of all three combinations under drought conditions grouped together with significantly higher oxidative stress values and notably lower growth performance. The second main cluster included well-irrigated applications represented by lower stress parameter values and higher averages of plant growth parameters and soluble protein content. Within this cluster, the 100 ppm TiO₂-NP treatments of 41 B/CS and 5 BB/CS under well-irrigated conditions formed the first subgroup, with lower growth performance. In the second subgroup, the 100 ppm TiO₂-NP treatment of 1103 P/CS under well-irrigated conditions was separated, while the 10 ppm TiO₂-NP treatments of all three combinations under well-irrigated conditions stood out, with significantly lower oxidative stress values and markedly higher growth performance.

4. Discussion

4.1. Characterization of TiO₂-NPs

The UV–vis analysis of TiO₂-NPs revealed a maximum absorption band at approximately 318 nm. In previous studies, TiO₂-NPs synthesized via green methods have been reported to show an absorption peak at around 350 nm [52]. The shift of the absorption peak to a lower wavelength or higher energy levels suggests variations in the NPs’ sizes [53]. XRD analysis indicated that the TiO₂-NPs possessed an anatase phase and a nanocrystalline structure. These findings align with various reports where TiO₂-NPs were synthesized using different plant extracts [54,55]. SEM analysis revealed that the TiO₂-NPs were spherical and exhibited a heterogeneous size distribution ranging from 16 to 23 nm. Consistent with our findings, Ghomrasni et al. [56] reported that the TiO₂-NPs they synthesized had an average SEM particle size of 21.1 nm. The EDX analysis of TiO₂-NPs indicated that the synthesized sample contained 66.3% titanium and 31.8% oxygen. Similar elemental compositions of titanium and oxygen have been reported in TiO₂-NPs synthesized using the green synthesis method with Syzygium cumini extract [57].

4.2. Effects of TiO₂-NPs on the Growth Characteristics of Grapevine Saplings

According to our results, drought stress significantly reduced plant growth parameters, including leaf area, shoot length, leaf number, root length, shoot dry and fresh weight, and root dry and fresh weight (Figure 2A–F and Figure 3A–D). However, the root dry matter ratio was not significantly affected by drought stress. Treatment with TiO₂-NPs, particularly at a concentration of 10 ppm, resulted in significant improvements in leaf number, leaf area, shoot fresh and dry weight, shoot length, root length, and root dry weight under both drought and well-irrigated conditions. These improvements suggest that TiO₂-NPs can enhance plant growth by mitigating the adverse effects of drought stress. Previous studies have indeed shown that TiO₂-NPs can increase nutrient absorption, particularly magnesium and nitrogen, which are critical for chlorophyll content and photosynthetic efficiency [58]. Our findings align with this, as the observed increase in leaf area and shoot growth under drought stress indicates improved photosynthetic performance and nutrient uptake. Similarly, the enhancement in root growth observed in our study supports literature reports that TiO₂-NPs can expand root pores and promote water and nutrient absorption, leading to better overall plant growth [59]. TiO₂-NP applications also improved the root dry matter ratio under drought stress, particularly at lower concentrations. This result is consistent with studies reporting that TiO₂-NPs can stimulate cell elongation and growth under stress conditions by loosening the cell wall and promoting cellular expansion [33]. Additionally, the observed increase in the shoot dry matter ratio under drought conditions suggests that TiO₂-NPs may mimic plant growth hormones such as cytokinin and gibberellin, thereby enhancing growth despite environmental stressors [60]. Our findings further confirm that TiO₂-NP treatments can enhance shoot and root biomass, as observed in studies on other crops such as barley, spinach, and mung bean [20,61,62]. The significant increase in both fresh and dry biomass under well-irrigated and drought conditions is indicative of TiO₂-NPs acting as a nanofertilizer, boosting metabolic activity and growth [63]. Similarly, the positive impact of TiO₂-NPs on root and shoot growth in our study aligns with earlier reports on Zea mays and other crops, highlighting the broad applicability of these nanoparticles in enhancing plant growth across different species [64,65]. In general, the positive effects of TiO₂-NPs on plant growth, particularly under drought conditions, can be attributed to their ability to enhance nutrient and water uptake, mitigate oxidative stress, and possibly mimic plant growth hormones. The consistency of these findings with the literature reinforces the potential of TiO₂-NPs as a valuable tool in improving plants’ resilience to environmental stressors and promoting sustainable agriculture.

4.3. Effects of TiO₂-NPs on Some Physiological and Biochemical Properties

Under drought conditions, leaf temperature, drought index, EL, and H₂O₂ and MDA levels increased significantly (Figure 4B–F). The application of 1 and 10 ppm TiO₂-NPs notably reduced these stress markers, while under well-irrigated conditions, higher TiO₂-NP concentrations led to an increase in these parameters. Additionally, the drought index decreased significantly under drought conditions with the application of 1 and 10 ppm TiO₂-NPs. ROS generated by drought stress can damage cell membranes and activate pectinases, leading to the degradation of pectin, a crucial component of cell walls. This degradation weakens the cell wall integrity, causing increased electrolyte leakage as ions are released from the cells [66,67]. The reduction in EL observed with the TiO₂-NP applications can be attributed to the nanoparticles’ ability to reduce ROS production and improve membrane integrity, thereby mitigating ROS-induced membrane damage. Similar results were reported by Mohammadi et al. [68], who demonstrated that TiO₂-NPs alleviated membrane damage in chickpea plants under cold stress. MDA and H₂O₂ are recognized as key indicators of drought-induced oxidative stress [69]. Polyunsaturated fatty acids, major lipid components of membranes, are particularly susceptible to peroxidation by ROS [20]. ROS attack unsaturated fatty acids in cellular membranes, initiating lipid peroxidation, which results in the breakdown of lipids and the formation of MDA as a byproduct [70]. MDA is commonly used as a biomarker to assess oxidative damage through lipid peroxidation. Plants respond to drought-induced oxidative stress by activating their antioxidant defense systems, which involve enzymes that protect against lipid peroxidation and scavenge ROS. However, under severe and prolonged drought stress, the antioxidant defense mechanism can become overwhelmed, leading to elevated MDA values [71].

Studies have shown that both metallic and non-metallic nanoparticles may stimulate the production and activity of antioxidant enzymes, thereby enhancing the antioxidant defense system in plants [20]. TiO₂-NPs, in particular, have been reported to reduce the formation of lipid radicals and suppress the propagation of lipid peroxidation chain reactions, ultimately contributing to lower MDA levels [72]. However, reports on the effects of TiO₂-NPs have been inconsistent. For instance, some studies have reported growth inhibition and increased lipid peroxidation in plants such as Nitzschia closterium treated with TiO₂-NPs [73], while others have observed toxic effects in onion plants at concentrations of 319 ppm [74]. These discrepancies can be attributed to differences in the plant species, nanoparticle sources, concentrations, and the specific factors evaluated. In general, research has suggested that TiO₂-NPs can have beneficial effects on plants’ physiological performance at low concentrations, whereas higher concentrations may lead to phytotoxicity [75,76]. Drought conditions led to a significant increase in total phenolic content and proline levels (Figure 5A,B). The application of 1 and 10 ppm TiO₂-NPs reduced total phenolic content and proline levels under drought conditions, while higher TiO₂-NP concentrations under well-irrigated conditions caused significant increases in these parameters. The accumulation of osmolytes such as proline is a well-established adaptive mechanism in plants under stress conditions. Proline acts as a compatible solute, playing a key role in osmotic regulation in many stressed plants. Its accumulation may result from the activation of proline synthesis through the glutamate pathway. Proline buildup is a widely recognized response to various types of stress, including drought [77,78,79]. In agreement with our findings, studies have reported that proline content doubles in plants exposed to drought stress, whereas plants treated with TiO₂-NPs show a much lower increase in proline levels compared with the controls [20]. The increased proline accumulation observed in plants exposed to nanoparticles may be part of the plants’ defense mechanism to protect cellular structures from damage caused by excessive ROS and MDA formation [80]. Secondary metabolite production in plants is largely dependent on the growth conditions, and stress has been found to significantly affect the cycles responsible for the storage and synthesis of secondary metabolites [81].

Nanoparticles exhibit different functions depending on their shape, size, concentration, experimental conditions, plant species, and uptake mechanisms [82]. Nanomaterials may be utilized as plant growth regulators to promote the production of secondary metabolites [83]. TiO₂-NP-treated plants have shown significant increases in total phenolic and flavonoid content compared with the controls [20]. Kamalizadeh et al. [84] reported that low concentrations of TiO₂-NPs increased the production of chlorogenic acid and rosmarinic acid in Dracocephalum moldavica. The pleiotropic effects of TiO₂-NPs on the synthesis of phenolic compounds followed a concentration-dependent pattern [20]. Drought stress caused a significant reduction in protein content (Figure 5C). However, TiO₂-NP applications, particularly at 1 and 10 ppm, were effective in increasing protein content under both drought and well-irrigated conditions. Consistent with our findings, significant increases in protein content have been observed in plants treated with TiO₂-NPs compared with the controls [20]. Antioxidant enzyme activities, including SOD, CAT, and APX, increased significantly under drought stress (Figure 5D–F). However, the application of 1 and 10 ppm TiO₂-NPs resulted in a significant reduction in these enzyme activities under drought conditions. Conversely, higher Se concentrations under well-irrigated conditions increased CAT and SOD activities, while APX activity was not significantly affected by TiO₂-NP treatments. Oxidative stress typically triggers the activation of antioxidants [82]. The decrease in enzyme activity may be related to increased H₂O₂ production [20]. SOD plays a crucial role in the initial stages of oxidative reactions by converting superoxide radicals into H₂O₂. Peroxidase then neutralizes hydrogen peroxide and contributes to the construction, strengthening, and eventual lignification of plant cell walls, helping to protect plant tissues from damage [77]. TiO₂-NPs have been reported to enhance water and nitrogen use efficiency and stimulate the activity of certain antioxidant enzymes in plants [85]. However, nanoparticles can also induce ROS in plants, contributing to oxidative stress [86]. The increase in phenolic compounds and antioxidant capacity observed in TiO₂-NP-treated plants may play a key role in scavenging ROS, thereby mitigating oxidative damage.

4.4. General Evolution

The results of the correlation analysis elucidated the intricate relationships among some plant growth, physiological, and biochemical parameters (Figure 6). These findings enhance our understanding of how different irrigation regimes, TiO₂-NP concentrations, and rootstock/cultivar combinations impact the parameters studied in grapevine saplings. Strong positive correlations were observed among vine growth parameters. For instance, the high positive correlation between shoot fresh weight and shoot dry weight suggests a direct relationship between fresh and dry forms of plant biomass, indicating that an increase in fresh biomass is accompanied by an increase in dry weight post-drying [58]. Similarly, the robust positive correlation between shoot length and leaf area indicates that longer shoots are associated with greater leaf area, potentially reflecting increased photosynthetic capacity [33]. Conversely, one of the strongest negative correlations among the vine growth parameters was observed between shoot length and drought index. This negative relationship implies that shoot elongation significantly decreases under drought stress, which can be explained by the plant’s strategy to limit growth to minimize water loss during drought conditions [87]. Correlations were also found among the physiological parameters. The strong positive correlation between electrolyte leakage and leaf temperature indicates that increases in stress conditions lead to a disruption of cellular membrane integrity, resulting in elevated leaf temperature [66]. The positive correlation between stomatal conductance and the SPAD index suggests that open stomata are associated with a higher chlorophyll content, which is indicative of enhanced photosynthetic activity [88]. On the other hand, the negative correlation between electrolyte leakage and RWC shows the direct relationship between water status in plant tissues and membrane integrity [89]. Additionally, the negative correlation between leaf temperature and RWC indicates that reduced water content leads to an increase in leaf temperature, likely due to reduced transpiration as plants close their stomata to conserve water [90].

Biochemical parameters also displayed significant correlations. The positive correlation between total phenolics and hydrogen peroxide levels suggests that increased ROS in oxidative stress conditions stimulates the accumulation of phenolic substances as part of the plant’s defense mechanisms [20]. Conversely, the negative correlation between hydrogen peroxide levels and soluble protein content implies that oxidative stress adversely affects protein structures, potentially leading to a reduction in protein levels under stress conditions [72]. The relationships among plant growth characteristics and biochemical and physiological parameters provide profound insights into plants’ responses to stress conditions. For instance, the strong positive correlation between RWC and shoot length indicates that higher water content is associated with better growth performance. Conversely, the negative correlation between electrolyte leakage and shoot length highlights that membrane damage under stress conditions negatively impacts plant growth [68]. PCA elucidated the effects of TiO₂-NP applications on drought stress tolerance and plant growth (Figure 7). The findings suggest that the analyzed parameters can be effectively explained through component analysis, indicating that TiO₂-NP applications may modulate stress responses and influence growth parameters differently. Notably, the application of TiO₂-NPs at a 10 ppm concentration enhanced plant growth under both well-irrigated and drought conditions. However, the 1 and 100 ppm concentrations exhibited varying effects depending on the irrigation regime. These findings suggest that TiO₂-NPs could play a potentially significant role in grapevine cultivation, depending on the concentration applied. Similar studies have reported that TiO₂-NP applications modulated stress responses and promoted growth in maize [64] and Verbascum sinuatum [20]. Hierarchical clustering heat map analysis supports the PCA’s findings, indicating that 10 ppm TiO₂-NP applications provided superior performance in the growth characteristics and physiological parameters, as well as a reduction in stress damage across all rootstock/cultivar combinations (Figure 8). The data suggest that high concentrations of TiO₂-NPs, when combined with drought stress conditions, might exacerbate stress damage rather than mitigate it. Conversely, lower TiO₂-NP concentrations demonstrated potential for improving drought damage compared with control samples, with higher growth values and lower oxidative stress parameters. This indicates that TiO₂-NP applications could potentially stimulate plant growth and alleviate stress responses within specific concentration ranges. The literature also supports these findings, with studies showing TiO₂-NP applications modulating stress responses and enhancing growth in plants [63,72,91].

5. Conclusions

The results of this study revealed that foliar application of TiO₂-NPs effectively mitigated oxidative damage in grapevine saplings by modulating the antioxidant defense mechanisms, including the activities of CAT, SOD, and APX enzymes, as well as total phenolic content. This intervention balanced proline and protein accumulation while simultaneously reducing the indices of lipid peroxidation, membrane damage, and H₂O₂ production. These effects collectively contributed to the alleviation of oxidative stress. Moreover, TiO₂-NP applications improved photosynthetic activity; RWC, reflected by the SPAD index and SC; and various growth parameters, thereby enhancing the drought tolerance of the grapevines. The study identified that the application of TiO₂-NPs at a concentration of 10 ppm exhibited the most significant positive impact on mitigating the effects of stress under drought conditions. In contrast, the effects of the 1 ppm TiO₂-NP concentration were limited, and the 100 ppm concentration resulted in phytotoxic effects, particularly under drought stress. These findings suggest that TiO₂-NPs have the potential to serve as effective elicitors for reducing abiotic stress, provided they are applied within the optimal concentration ranges. This approach represents a promising avenue for addressing drought issues in viticulture and exploring semi-arid agricultural practices. However, it is imperative to conduct comprehensive safety assessments, including the evaluation of potential impacts on non-target organisms, prior to the application of nanomaterials in agricultural settings. Future research should focus on a more detailed assessment of the effects of TiO₂-NP applications across different irrigation regimes and plant species. Furthermore, elucidating the molecular mechanisms through which TiO₂-NPs influence plants’ growth and stress tolerance will be essential for optimizing their use in agricultural practices.