Foliar Titanium Dioxide Nanoparticles Enhance Rice Yield by Improving Photosynthesis, Ion Balance, and Antioxidant Defense Under Salt Stress

TL;DR

Spraying rice plants with titanium dioxide nanoparticles improves their growth and yield under salt stress by boosting photosynthesis, ion balance, and antioxidant defenses.

Contribution

This study reveals the effectiveness of nano-TiO2 in enhancing rice productivity under salinity through physiological and genetic mechanisms.

Findings

Ti2 and Ti3 treatments increased grain yield by 8.59% and 14.80% in salt-tolerant and salt-sensitive rice varieties.

Nano-TiO2 improved photosynthesis, antioxidant enzyme activity, and nitrogen metabolism in rice under salt stress.

Application of nano-TiO2 upregulated ion transport-related genes, optimizing the K+/Na+ ratio in rice leaves.

Abstract

Salinity stress severely limits rice productivity and grain quality worldwide. Although exogenous foliar application of titanium dioxide nanoparticles (nano-TiO2) has been reported to enhance crop stress tolerance, its regulatory roles in yield formation and grain quality in rice varieties with differing salt tolerance are not well understood. In the present study, two contrasting rice varieties, viz., Jingliangyou 3261 (JLY3261; salt-tolerant) and Yuxiangyouzhan (YXYZ; salt-sensitive), were applied with five nano-TiO2 foliar application treatments—viz., CK: water spray; Ti1: 15 mg L−1; Ti2: 30 mg L−1; Ti3: 45 mg L−1; and Ti4: 60 mg L−1—at the jointing and panicle initiation stages. Plants were irrigated with 0.3% saltwater to simulate salt stress. The results showed that Ti2 and Ti3 treatments led to 8.59% and 14.80% increases in grain yield in JLY3261 and YXYZ, respectively, compared with CK. Ti2 and Ti3 treatments significantly increased the leaf area index, net photosynthetic rate, and aboveground biomass of both varieties at the heading stage. Meanwhile, the activities of antioxidant enzymes such as superoxide dismutase and peroxidase, as well as nitrogen metabolism enzymes including nitrate reductase and glutamine synthetase, were improved with a substantial reduction in malondialdehyde contents. Application of nano-TiO2 upregulated the expression of ion transport-related genes such as OsSOSs, OsNHXs and OsHKTs, thus improving leaf K+ accumulation and reducing Na+ content to optimize the K+/Na+ ratio. In addition, Ti2 and Ti3 treatments improved the milled rice rate, head rice rate, and protein content, while they decreased the chalkiness degree of both rice cultivars. Principal component analysis showed that the aboveground biomass at the heading stage was a core evaluation index for both varieties. Overall, foliar application of 30–45 mg L−1 nano-TiO2 was found to be effective regarding growth and yield improvement in rice under saline conditions. This study provides a theoretical basis for agro-management strategies for rice cultivation in saline–alkaline soils.

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1. Introduction

Nearly one and half billion hectares of land worldwide have been affected by salinity [1]. The area of saline land is continuously expanding at a rate of one million hectares per year through natural and anthropogenic activities [2]. Excessive salt in the soil not only damages soil structure and reduces nutrient availability, but also imposes multiple stresses on crop growth, making it one of the key non-biological factors restricting the crop productivity of the agricultural systems of China [3].

With population growth and the reduction in arable land resources, developing rice (Oryza sativa L.) production by utilizing saline–alkali land and ensuring national food security has become an important approach in China [4,5]. However, rice is highly sensitive to salt stress, and salt stress causes systemic damage to rice through oxidative stress, osmotic stress and ion toxicity [6]. At the ionic level, excessive Na^+^ competes with K^+^ and other essential mineral elements for absorption and transport, disrupting the intracellular ion balance and normal metabolic activities [7]. At the osmotic level, high salt concentrations cause cell dehydration and wilting, inhibit root water absorption and nutrient transportation, and hinder plant growth and development [1]. At the oxidative level, salt stress triggers the outbreak of reactive oxygen species (ROS), causing lipid peroxidation (in terms of malondialdehyde content (MDA) accumulation) and degradation of chlorophyll [8]. Ultimately, it leads to a reduction in the number of effective panicles, rate of grain formation, grain weight, and yield [9]. Meanwhile, salt stress also deteriorates rice grain quality with a substantial reduction in brown rice and polished rice and an increase in chalkiness degree and abnormal amylose content, which adversely reduces the commercial value and edible quality of rice [10].

Traditional approaches to alleviate salt stress in rice mainly involve water management (e.g., salt-free irrigation), chemical amendments (e.g., gypsum and organic fertilizers), and breeding of salt-tolerant varieties [1]. Water improvement can quickly reduce soil salinity, but it has the drawbacks of high water resource consumption, high cost, and the potential for secondary salinization [11]. Chemical improvement can temporarily improve the physicochemical properties of the soil, but long-term application may lead to soil pollution and nutrient imbalance [12]. Breeding of salt-tolerant varieties is the fundamental approach, but it is associated with a long breeding cycle, a complex genetic background, and difficulty in balancing salt tolerance and high-quality high-yield traits [13]. The limitations of these traditional strategies highlight the urgent need for efficient, eco-friendly, and practical approaches to mitigate salt stress.

The rapid development of nanotechnology has provided an innovative direction for managing abiotic stresses in agricultural systems [14]. Nano-titanium dioxide (nano-TiO_2_) exhibits significant advantages in alleviating salt stress in crops, owing to its unique physicochemical properties and environmental compatibility [15]. Nano-TiO_2_ presents multiple advantages over traditional measures, including excellent biocompatibility, low toxicity at effective concentrations, and minimal risk of environmental footprints [16]. Nano-TiO_2_ has been proven to exert stress-mitigating effects in a variety of plant species, and its regulatory efficiency is closely related to the plant species, stress type, application concentration and administration method [15,17,18]. In soybean, foliar application or seed priming with nano-TiO_2_ significantly attenuated salt stress damage by improving metabolic constituents and cell ultrastructure, and increased seedling vigor and photosynthetic performance [17,19]. In maize, nano-TiO_2_ seed priming alleviated nickel-induced stress by enhancing antioxidant defense and optimizing cell ultrastructure, and improved plant biomass and nutrient uptake [15]. Mustafa et al. [20] found that foliar applications of plant-based titanium dioxide nanoparticles improved root and shoot fresh and dry weight and total chlorophyll contents of wheat (Triticum aestivum L.) plants under salinity stress.

However, the concentration-dependent effects and regulatory thresholds of nano-TiO_2_ remain unclear, and comprehensive studies on the systemic impact of foliar application on rice yield and grain quality are lacking. Therefore, the present study was conducted to assess the effects of foliar-applied nano-TiO_2_ on rice under salt stress, focusing on growth, yield, physiological and biochemical responses, ion homeostasis, and grain quality, as well as to elucidate the molecular regulation of Na^+^-K^+^ transporter genes and to identify optimal application concentrations for alleviating salt stress in rice grown in saline–alkaline soils.

2. Materials and Methods

A pot experiment was conducted in Jiusuo Village, Hainan Province (108.89° E, 18.45° N), during 2024. This area has a typical tropical maritime monsoon climate. Two rice varieties, i.e., Jinliangyou 3261 (JLY3261), salt-tolerant, and Yuxiangyouzhan (YXYZ), salt-sensitive, were placed in pots filled with loamy soil with 2.45 g kg^−1^ total nitrogen, 245.13 mg kg^−1^ available nitrogen, 81.57 mg kg^−1^ available phosphorus, and 158.55 mg kg^−1^ available potassium.

2.1. Plant Material and Design

The seedlings were then transplanted at the three-leaf stage (25 days after sowing), with two plants per hill and three hills per pot. Each pot (36 cm top diameter; 26 cm bottom diameter; and 28 cm in height) was filled with 15 kg of dry soil. The experimental treatments included water spray (CK) and four levels of nano-TiO_2_, viz., 15 mg L^−1^ (Ti1), 30 mg L^−1^ (Ti2), 45 mg L^−1^ (Ti3), and 60 mg L^−1^ (Ti4), which were sprayed at jointing and the spike emergence stage. For each concentration treatment, nano-TiO_2_ powder was accurately weighed with a 0.1 mg precision analytical balance (15 mg, 30 mg, 45 mg, and 60 mg for Ti1 to Ti4, respectively). The powder was first pre-dispersed in 50 mL of ultrapure water to form a uniform paste, and then fully transferred to a 1 L volumetric flask. The beaker and glass rod were rinsed with ultrapure water more than 3 times to ensure complete transfer of the powder, and the volume was fixed to 1 L with ultrapure water. The pots were arranged in a completely random design with 10 pots per treatment. All treatments were irrigated with 0.3% saltwater to simulate salt stress. A detailed introduction to TiO_2_ is provided.

The ZnO NPs were a commercial product and were purchased from Shanghai Maclin Biochemical Technology Co., Ltd. (Shanghai, China). The specifications of nano-TiO_2_ are: 99% metal basis purity, 20–40 nm particle size, anatase crystal type, hydrophilic surface property, 70–85 m^2^ g^−1^ specific surface area, and white fluffy powder. The TiO_2_ suspension was freshly prepared immediately before each foliar spraying, strictly in accordance with the mature and widely verified method described in previous authoritative studies on foliar application of nano-TiO_2_ [14,20]. To prepare the foliar spraying suspension, we dispersed the powder in ultrapure water without adding any other compounds (e.g., dispersants, surfactants, or stabilizers), and the mixture was subjected to water bath sonication to achieve uniform dispersion of the particles with no obvious agglomeration. Since the suspension was composed solely of nano-TiO_2_ and ultrapure water, there were no additional compounds that could potentially impact plant health, and thus, no additional control experiments for such compounds were required.

During the seedling establishment stage, freshwater was used for irrigation. Twenty days after transplanting, saline irrigation was initiated using 0.3% seawater. Saline water was prepared by mixing seawater and groundwater with a water pump to achieve the target concentration. Irrigation was suspended once the root-zone soil salinity reached 0.3%. Soil salinity was monitored using an electrical conductivity meter (2266FS, Spectrum, Boston, MA, USA), and saline irrigation was resumed whenever salinity fell below 0.3% to maintain the target level. Nitrogen (N), phosphorus (P), and potassium (K) fertilizers were applied at equivalent rates of 2.76 g urea pot^−1^, 3.03 g calcium superphosphate pot^−1^, and 1.18 g potassium chloride pot^−1^, respectively. Nitrogen was applied in the form of urea (46% N) in three splits, i.e., basal, tillering, and panicle initiation, at a ratio of 1:1:1. Phosphorus was applied as calcium superphosphate (14% P) at a rate equivalent to 60 kg P ha^−1^ and applied as basal fertilizer. Potassium was applied as potassium chloride (60% K) at a rate equivalent to 100 kg K ha^−1^ and split equally between basal and panicle initiation stages (1:1).

2.2. Sampling and Measurements

2.2.1. Determination of Yield and Its Components

At maturity, the yield and its components were determined according to Li et al. [21]. Three pots per replicate were sampled to estimate yield and yield components; i.e., productive panicles per pot, spikelets per panicle and seed-setting rate (filled grains/ total spikelets × 100) were recorded. The grains were oven-dried to 13.5% moisture, and 1000-grain weight was determined. Three pots were harvested from each treatment, sun-dried, and weighed to obtain the grain yield.

2.2.2. Determination of Dry Matter Accumulation and Translocation

At both heading and maturity, the dry matter accumulation and translocation were determined according to Li et al. [21]. The six uniform plants per treatment were separated into stem–sheath, leaf and panicle (post-heading) organs, dried at 105 °C for 30 min, and then dried at 80 °C to constant weight to estimate dry biomass. Total dry matter accumulation, translocation amount and efficiency were calculated using the following: translocation amount = dry mass of vegetative organs (stem–sheath + leaf) at heading − dry mass of vegetative organs at maturity.

2.2.3. Leaf Area Index (LAI) and Relative LAI Decline

At heading and maturity, the total green leaf area of each plot was measured. The LAI was calculated as total leaf area divided by ground area. The relative decline in the leaf area index (LAI) from heading to maturity was quantified using the LAI at heading as the reference peak: (LAI_heading − LAI_maturity)/LAI_heading.

2.2.4. Measurement of Photosynthetic Characteristics

The flag leaves of plants in each treatment were selected at the heading stage, and photosynthesis was measured during 9:00–11:00 a.m. The net photosynthetic rate (P_n_), stomatal conductance (G_s_), intercellular CO_2_ concentration (C_i_), and transpiration rate (T_r_) were measured using a portable photosynthesis instrument according to Wei et al. [22].

2.2.5. Determination of Sodium (Na+) and Potassium Ion (K+) Contents

The Na^+^ and K^+^ contents were determined according to Jin et al. [23]. Dried leaves were ground and passed through a 40-mesh sieve. Then, 0.3 g of the powder was microwave-digested using H_2_O_2_-H_2_SO_4_. The Na^+^ and K^+^ contents were estimated using an inductively coupled plasma atomic emission spectrometer (ICP-OES) (IRIS Intrepid II XSP, Thermo, Massachusetts, USA).

2.2.6. Expression of Na+/K+ Transport Genes

Representative Na^+^ and K^+^ transport genes identified from prior RNA-seq analysis were quantified by qRT-PCR to assess transcript-level responses across stress intensities and durations (Table S1). All primers are listed in Supplementary Table S2. The 2^−ΔΔCT^ method, as described by Chen et al. [24], was used to calculate the relative gene expression levels using the mean value of four replicates.

2.2.7. Determination of Antioxidant Enzyme Activity, Nitrogen Metabolism Enzyme Activity, and Malondialdehyde (MDA) Content

The antioxidant enzyme activity, nitrogen metabolism enzyme activity, and MDA were determined according Zhao et al. [25]. The activities of superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), nitrate reductase (NR), glutamine synthetase (GS), and glutamate synthase (GOGAT) and the content of MDA were determined using kits purchased from Jiangsu Jingmei Biotechnology Co., Ltd. China (Jiangsu, China). The detailed measurement method can be found in Supplementary Material S1.

2.2.8. Determination of Reactive Oxygen Species (ROS) Content

Fresh leaves (0.5 g), collected at heading stage, were homogenized with 5 mL of pre-cooled 5% trichloroacetic acid solution. The sample was ground on ice, and then centrifuged at 4 °C and 12000 rpm for 20 min. The rate of superoxide anion (O_2_^−^·) production was determined by the nitroblue tetrazolium method, and the content of hydrogen peroxide (H_2_O_2_) was determined by the titanium salt colorimetric method.

2.2.9. Grain Quality Assessment

After harvest, grains were oven-dried to a moisture content of 13.5%. Processing quality traits, including brown rice, milled rice, and head rice percentages, as well as appearance quality (chalky grain rate), were determined following the methods described by Jin et al. [23]. Protein content was determined according to Jin et al. [26].

2.3. Data Analysis

Data processing was performed with Excel 2021 and analysis was conducted by statistical software SPSS 26.0 (SPSS Inc., Chicago, IL, USA) using the two-way variance (ANOVA) technique and principal component analysis (PCA). A two-way analysis of variance (two-way ANOVA) was employed to evaluate the independent and interactive effects of two fixed factors: rice variety and nano-TiO_2_ concentration. Significant differences were calculated based on the least significant difference (LSD) test at the p < 0.05 level. Graphs were drawn using Origin 2023 (OriginLab Corp., Northampton, MA, USA).

3. Results

3.1. Grain Yield and Its Components

Exogenous application of nano-TiO_2_ regulated the grain yield and its components for two rice varieties under salt stress (Table 1). In JLY3261, grain yield and its components initially increased and then declined with increasing nano-TiO_2_ concentrations, with the optimal regulatory effect observed under the Ti2 treatment. In JLY3261, the grain yield, spikelets per panicle, and 1000-grain weight were increased by 8.59%, 5.37%, and 4.10%, respectively, in Ti3 treatment, as compared with CK. Moreover, the grain yield of YXYZ in Ti3 treatment was 14.80% higher than that of CK, and the spikelets per panicle, grain filling, and 1000-grain weight were also increased by 4.79%, 9.56%, and 4.25%, compared with CK. For YXYZ, the grain yield under Ti4 was 4.13% higher than CK, and no significant differences were observed in effective panicles per plant or spikelets per panicle compared with CK. Both the cultivar and nano-TiO_2_ factors significantly affected the yield, grain filling, and spikelets per panicle. The cultivar × nano-TiO_2_ interaction remained not significant for grain yield and its components.

3.2. Leaf and Stem–Sheath Weight Accumulation and Translocation

Exogenous nano-TiO_2_ application substantially affected dry matter accumulation at heading and dry matter redistribution at maturity in both rice varieties under salt stress (Table 2). In YXYZ, the maximum dry matter accumulation occurred under the Ti3 treatment, with leaf and stem–sheath dry matter reaching 24.95 and 8.77 g plant^−1^, respectively, representing increases of 32.01% and 17.25% over CK. In contrast, JLY3261 showed optimal regulation in the Ti2 treatment, where leaf and stem–sheath dry matter reached 23.91 and 8.70 g plant^−1^, corresponding to increases of 13.73% and 22.30% compared with CK. Moreover, dry matter translocation efficiency at maturity was consistently higher in JLY3261 than in YXYZ in the same nano-TiO_2_ treatment. Both the cultivar and nano-TiO_2_ factors significantly affected the stem–sheath weight and stem–sheath translocation. The cultivar × nano-TiO_2_ interaction remained significant for stem–sheath weight at maturity, leaf translocation, and stem–sheath translocation.

3.3. Leaf Area Index (LAI) and Aboveground Accumulation

For JLY3261, the maximum LAI was observed under the Ti2 treatment, which was significantly higher than CK, whereas no significant difference was found between Ti2 and Ti3 treatments in the LAI during the heading and maturity stages. In YXYZ, the optimal regulatory concentration of nano-TiO_2_ was Ti2, under which the LAI peaked, representing an increase of 11.8% and 57.4% at heading and maturity, respectively, compared with CK (Figure 1).

Regarding aboveground biomass, Ti2 and Ti3 were found to be comparatively effective for JLY3261 and YXYZ, respectively, at the heading stage (Figure 1). At the maturity stage, the total dry matter accumulation of both varieties was higher than that at the heading stage. The maximum total dry biomass of JLY3261 was recorded under Ti2 treatment, which was 11.99% higher than CK. For YXYZ, no significant difference was found between nano-TiO_2_ treatments; however, the total dry biomass in all nano-TiO_2_ remained comparatively higher than CK. Both the cultivar and nano-TiO_2_ factors significantly affected the LAI at maturity and the relative decline rate of the LAI. The cultivar × nano-TiO_2_ interaction was significant for the relative decline rate of the LAI (Table S3).

3.4. Photosynthetic Characteristics

For JLY3261, the photosynthetic parameters such as P_n_, G_s_, C_i_, and T_r_ responded positively to nano-TiO_2_, followed by a decline in the Ti4 treatment. The Ti2 treatment resulted in the most pronounced enhancement of photosynthetic performance, with P_n_, G_s_, and T_r_ increasing by 12.50%, 15.24%, and 34.59% compared with CK. Meanwhile, C_i_ in JLY3261 under Ti3 treatment was decreased by 9.71% compared with CK. For YXYZ, maximum P_n_ was recorded under Ti2 treatment, i.e., 17.40% higher than CK. Correspondingly, the C_i_ under Ti3 treatment dropped to 13.70% lower than CK in YXYZ. Moreover, the P_n_, G_s_, and T_r_ under Ti4 were 5.14%, 11.18%, and 18.75% higher, respectively, than CK (Figure 2). Both the cultivar and nano-TiO_2_ factors significantly affected the P_n_, G_s_, C_i_, and T_r_ (Table S3).

3.5. Leaf Sodium (Na+) and Potassium Ion (K+) Content and K+/Na+ Ratio

For JLY3261, leaf Na^+^ content showed a continuous decreasing trend with an increase in nano-TiO_2_ concentration from 0 to 30 mg L^−1^, while leaf K^+^ content and the K^+^/Na^+^ ratio presented an opposite increasing trend. For YXYZ, the lowest leaf Na^+^ content and the highest K^+^ contents were recorded under the Ti3 treatment, with the highest K^+^/Na^+^ ratio (Figure 3). Both the cultivar and nano-TiO_2_ factors significantly affected the Na^+^, K^+^, and K^+^/Na^+^ ratio (Table S3).

3.6. Relative Expression Levels of Na+ and K+ Transport-Related Genes

Exogenous nano-TiO_2_ application exerted a distinct regulatory effect on the transcription levels of ion homeostasis-related gene families, including OsSOS (OsSOS1/2/3), OsHKT (OsHKT1;1/1;3/1;5) and OsNHX (OsNHX1/2/3), in both rice varieties under salt stress (Figure 4). Among the OsSOS family, the highest OsSOS1, OsSOS2, and OsSOS3 relative expression under nano-TiO_2_ treatments was significantly higher than CK. In the OsHKT family, OsHKT1;5 showed the most pronounced upregulation under Ti2 treatment, with a relative expression level 109.00% higher than CK, whereas the upregulation of OsHKT1;1 and OsHKT1;3 was relatively moderate (35.24% and 80.39%, respectively). For the OsNHX family, OsNHX2 was highly induced by Ti4 treatment, with relative expression levels 54.90% higher than those of CK, while OsNHX1 showed a relatively weak response.

3.7. Antioxidase Enzyme Activity and Reactive Oxygen Species (ROS)

For JLY3261, the activities of SOD, POD, and CAT in CK were the lowest among all nano-TiO_2_ treatments. The Ti3 treatment resulted in maximum activities of SOD, POD, and CAT, which were 201.16%, 143.81%, and 56.70% higher than CK, respectively. The Ti2 treatment showed slightly lower enzyme activities than Ti3, but still significantly higher than CK and Ti1. In contrast, YXYZ had substantially lower SOD, POD, and CAT activities than those of JLY3261 under the same salt stress, as compared to CK. Exogenous nano-TiO_2_ induced smaller increases in antioxidant enzyme activities in YXYZ compared with JLY3261. In YXYZ, the optimal concentration was Ti2, which enhanced SOD, POD, and CAT activities by 64.62%, 132.76%, and 131.41%, respectively, relative to CK (Figure 5). Both the cultivar and nano-TiO_2_ factors significantly affected the SOD, POD, and CAT. The cultivar × nano-TiO_2_ interaction was significant for SOD, POD, and CAT (Table S3).

For JLY3261, the highest MDA, H_2_O_2_, and O_2_^−^· were detected in the CK, and nano-TiO_2_ treatments, especially Ti3, reduced MDA content by 25.7%, H_2_O_2_ content by 24.7%, and O_2_^−^· by 19.4% compared with CK (Figure 6). In YXYZ, nano-TiO_2_ application showed a more pronounced mitigation effect: the Ti2 treatment led to a 30.4% reduction in MDA and a 22.1% decrease in H_2_O_2_ relative to the corresponding CK values. Both the cultivar and nano-TiO_2_ factors significantly affected the MDA, H_2_O_2_, and O_2_^−^·. The cultivar × nano-TiO_2_ interaction was significant for MDA and O_2_^−^ (Table S3).

3.8. Nitrogen Metabolism Enzyme Activity

The flag leaf nitrogen metabolism enzyme activities, i.e., NR, GS, and GOGAT, during the heading stage are shown in Figure 7. For JLY3261, with the increase in nano-TiO_2_ concentration from Ti1 to Ti4, the activities of nitrogen metabolism enzymes were initially increased and then decreased. The Ti2 treatment achieved the maximum enzyme activities, with NR and GS activities being 17.71% and 47.96% higher than CK, respectively. The Ti1 treatment exhibited the highest GOGAT activity, while the Ti4 treatment only showed marginal improvements compared to CK. Moreover, exogenous nano-TiO_2_ application increased the nitrogen metabolism enzyme activities in YXYZ more than in JLY3261. The optimal concentration for YXYZ was Ti3 treatment, which led to 69.95% and 38.22% enhancement in GS and GOGAT activities, respectively, as compared with CK. Notably, the Ti4 treatment also improved the NR and GOGAT activities, but its promotive effect was comparatively lower than Ti3. Both the cultivar and nano-TiO_2_ factors significantly affected the NR, GS, and GOGAT. The cultivar × nano-TiO_2_ interaction was significant for GOGAT (Table S3).

3.9. Rice Grain Quality

Application of nano-TiO_2_ resulted in grain quality regulation, including processing, appearance, and nutritional quality (Table 3). With an increase in nano-TiO_2_ concentration from Ti1 to Ti4, the processing quality indices and protein contents were initially increased and then decreased, whereas the chalkiness degree exhibited an opposite trend. Compared with CK, the milled rice rate, head rice rate and grain protein contents were increased by 7.77%, 8.15%, and 8.26%, respectively, in Ti2, whereas the chalkiness degree was reduced by 27.62. The Ti3 treatment maintained relatively favorable quality traits, showing values significantly higher than CK and Ti1 but slightly lower than Ti2, whereas the Ti4 treatment resulted in only marginal improvements in processing quality and chalkiness degree and showed no significant difference in protein content compared with CK.

For YXYZ, the milled rice rate, head rice rate, and protein contents were increased by 5.89%, 9.97%, and 15.93% in Ti3 treatment, compared to CK. Notably, the Ti4 treatment also improved the rice grain quality. Nevertheless, its effectiveness was comparatively less than Ti2 and Ti3. The cultivar × nano-TiO_2_ interaction remained not significant for the chalkiness rate and protein content.

3.10. Principal Component Analysis (PCA)

For JLY3261, the PCA extracted two principal components (PC1 and PC2) with eigenvalues > 1, accounting for 51.1% and 11.8% of the total variance, respectively, with a cumulative variance contribution rate of 62.9%. This indicated that the two principal components could fully represent the original data information. PC1 was predominantly associated with positive loadings of aboveground biomass at heading (0.199), yield (loading = 0.198), and SOD (0.197), as well as RDLAI (−0.183). Treatment clustering in the PCA score plot showed distinct separation: Ti1, Ti3, and Ti4 were distributed between CK and Ti2, with Ti3 closer to Ti2 (Figure 8).

For YXYZ, two principal components were also extracted, with PC1 explaining 48.3% of the total variance and PC2 explaining 9.5%, achieving a cumulative contribution rate of 57.8%. PC1 for YXYZ was dominated by positive loadings of POD activity (0.214), the leaf K^+^/Na^+^ ratio (0.198), and aboveground biomass at heading (0.197), and negative loading of Na^+^ (–0. 167). Treatment clustering in the PCA score plot showed distinct separation; i.e., Ti1 and Ti4 were distributed between CK and Ti2, with Ti3 closer to Ti2 (Figure 8).

4. Discussion

Soil salinization is a global agricultural and ecological challenge that severely constrains sustainable agricultural development. It not only damages soil structure and reduces nutrient availability, but ultimately leads to a significant reduction in yield and grain quality [9,22]. Traditional measures for alleviating salt stress have many limitations, whereas advances in nanotechnology offer a promising alternative strategy. Among diverse nanomaterials, nano-TiO_2_, with its excellent biocompatibility and eco-friendly nature, shows great application potential in plant resilience under multiple abiotic stresses [16]. The present study identified variety-specific optimal concentrations and revealed the multi-pathway synergistic regulatory mechanism of nano-TiO_2_ in alleviating rice salt stress.

Salt stress directly damages the structure of chloroplasts, leading to a decrease in photosynthetic efficiency and subsequently affecting the synthesis and transport of dry matter [22]. Salt stress reduces the peak leaf area index and significantly suppresses P_n_ and G_s_ in rice, resulting in limited dry matter accumulation and reduced translocation efficiency [25]. Our research results also found that nano-TiO_2_ optimizes the distribution of dry matter between vegetative and reproductive organs, improves dry matter transport, and ensures that more photosynthetic products are transferred to grains, ultimately leading to yield improvement. This finding agrees with earlier reports that nano-ZnO promotes the transfer of photosynthetic products from vegetative tissues to grains under saline conditions [23]. The improvement in dry matter accumulation and translocation induced by nano-TiO_2_ can be well explained by its regulatory effects on the LAI and photosynthetic characteristics: nano-TiO_2_ significantly increased the LAI at both heading and maturity stages of rice, especially for the salt-sensitive variety, and delayed leaf senescence under salt stress, which expanded the effective photosynthetic area of rice and provided the structural basis for sufficient photosynthetic product synthesis. Abdelhameed et al. [17] found that nano-TiO_2_ application improved leaf pigmentation and protein content as well as P_n_. Consistent with these findings, the present study demonstrated that exogenous nano-TiO_2_ significantly enhanced P_n_ and G_s_, promoting CO_2_ absorption and assimilation, providing a solid foundation for dry matter accumulation. Alharbi et al. [27] found that nano-SiO_2_ enhanced the photosynthetic performance of crops under salt stress by promoting chlorophyll biosynthesis, increasing G_s_, and enhancing the photosynthetic efficiency of photosystem II. Rehman et al. [16] found that nano-TiO_2_ can simultaneously improve the electron transfer efficiency of PSII reaction centers and further strengthen photosynthetic performance. Liang et al. [28] found that nano-SiO_2_ also significantly increased the P_n_, G_s_, and T_r_ of cotton seedling leaves, and the length, width and density of stomata increased with the increase in nano-SiO_2_ concentration.

Ion homeostasis is a key physiological basis for salt stress tolerance in rice. Under salt stress, the excessive accumulation of Na^+^ competes with K^+^ for absorption and transport, disrupting the intracellular ion homeostasis and causing enzyme activity disorders and metabolic imbalances [29,30]. Our study found that nano-TiO_2_ optimized the leaf K^+^/Na^+^ ratio of rice under salt stress by reducing Na^+^ accumulation and enhancing K^+^ absorption in both varieties, which was attributed to the significant upregulation of K^+^ absorption gene OsHKT1 and downregulation of the overexpression of Na^+^ transport gene OsHKT1;5 and Na^+^ efflux gene OsSOS. The results were consistent with Alharbi et al. [27], who reported that nano-SiO_2_ enhances the ion selectivity to improve the Na^+^ and K^+^ homeostasis in barley leaves, which is consistent with findings from nano-TiO_2_-based stress resistance studies in other crops [31,32]. Li et al. [21] found that exogenous spraying of nano-ZnO is beneficial for increasing the K^+^/Na^+^ ratio in fragrant rice leaves and enhancing their stress resistance. In this study, nano-TiO_2_ treatment effectively improved the K^+^ and Na^+^ balance by regulating Na^+^ and K^+^ transport-related genes. Peng et al. [18] reported that the potassium ion transporter CsAKT1 is a key gene promoting K^+^ uptake in cucumber seedlings in response to nano-CeO_2_. Moreover, foliar application of nano-CeO_2_ induced CsAKT1 expression in leaves and roots more effectively than root application. Wang et al. [33] found that nano-SiO_2_ exogenous application is beneficial for increasing the K^+^ and Si contents in tomato seedlings and reducing the absorption of Na^+^. Liu et al. [34] found that foliar application of nano-CeO_2_ maintains the K^+^ retention and Na^+^ efflux by upregulating HKT1 and downregulating KOR, instead of Na^+^ vacuolar storage, maintaining the K^+^ and Na^+^ homeostasis in leaves and improving salt tolerance ability of cotton.

Salt stress can induce an over-production of ROS in plants [35]. Excessive oxidative stress that surpasses the antioxidant defense leads to membrane lipid peroxidation and biomolecular damage, markedly affecting rice growth and development [36,37]. Our study found that nano-TiO_2_ significantly increased the activities of key antioxidant enzymes (SOD, POD, CAT) in rice leaves and reduced malondialdehyde (MDA) content under salt stress, effectively alleviating oxidative damage. Abdalla et al. [38] found that nano-TiO_2_ has a positive effect on early growth by improving antioxidants to reduce H_2_O_2_ and MDA content. Nanoparticle-induced H_2_O_2_ acts as a warning signal, triggering the MAPK pathway and stress-responsive transcription factors (NAC, WRKY), which boost enzymatic and non-enzymatic antioxidants and strengthen cellular defenses against excessive ROS [39,40]. The results were consistent with Wang et al. [34], who found that nano-SiO_2_ can regulate the antioxidant enzyme system and remove excessive ROS under adverse conditions. Li et al. [21] found that exogenous nano-ZnO can effectively increase the activity of antioxidant enzymes and reduce the MDA content in leaves. Nano-TiO_2_ provides conditions for plant cells to resist oxidative stress as a nano-level redox regulator [16]. Exogenous nano-TiO_2_ significantly enhanced NR, GS, and GOGAT activities, optimizing nitrogen metabolism, supplying sufficient nitrogen for growth, and thereby improving rice stress tolerance and growth potential. This is consistent with the result of Omara et al. [41], who found that beneficial root-associated bacteria improve wheat productivity under salt stress by improving nitrogen metabolism. The antioxidant system and nitrogen metabolism synergy improvement jointly construct the physiological defense line of rice against salt stress. Ma et al. [42] found that methyl jasmonate improves the activity of key enzymes in plant carbon and nitrogen metabolism, increasing the content of carbohydrates and nitrogen-containing compounds to improve salt tolerance.

The formation of rice quality is the comprehensive result of the physiological metabolism throughout the entire growth period of rice. Under salt stress, the processing quality, appearance quality, and cooking flavor quality of rice undergo a comprehensive deterioration [10]. The nano-TiO_2_ effectively alleviates the deterioration effect of salt stress on rice quality through multi-pathway coordinated regulation. Nano-TiO_2_ application enhances photosynthesis and nitrogen metabolism, supplying essential nutrients for grain development, while alleviating ionic toxicity and oxidative stress to maintain metabolic stability. This ultimately improves processing and appearance quality, enhances cooking and eating traits, and increases the commercial value of rice. Jin et al. [23] found that nano-SiO_2_ can enhance rice quality under salt stress by improving physiological metabolism, whereas Ismail et al. [43] also reported that nano-SiO_2_ has an optimization effect on the quality of peas.

Notably, the alleviating effect of nano-TiO_2_ on salt stress was concentration-dependent. In our study, Ti2 and Ti3 were optimal for JLY3261 and YXYZ, respectively, increasing grain yield by 8.59% and 14.80% compared with CK, primarily due to enhanced spikelets per panicle, 1000-grain weight, and seed-setting rate. Similarly, Garg et al. [44] found that nano-Se improved rice yield under salt stress owing to an increase in spikelets. It was further noticed that yield benefits declined when nano-TiO_2_ was applied at concentrations exceeding 45 mg L^−1^. This is consistent with Rajput et al. [45], who reported that the optimal dosage of nanomaterials must be carefully controlled in practical applications. Excessive application tends to cause particle agglomeration, which reduces their activity, and may block stomata, thereby affecting gas exchange and transpiration rates [15,46]. Moreover, excessive nano-TiO_2_ may trigger oxidative stress, although low-concentration nano-TiO_2_ can activate the antioxidant system to remove ROS, but at high concentrations, it may lead to excessive accumulation of ROS, thus aggravating membrane lipid peroxidation damage (Figure 6). Therefore, optimal nanomaterial application depends on matching crop traits and stress levels and carefully controlling concentration to enhance stress resistance and yield without inducing negative effects. The efficacy of nanomaterials also depends on the application method. Undoubtedly, foliar spraying was found to be effective in this study; nevertheless, its application to the seed, soil, or root and crop stage, i.e., the panicle emergence period and filling period, may further improve its efficacy [47,48]. Simultaneously, application strategies should consider varietal salt tolerance to optimize concentration and timing, enabling a precise and effective salt stress mitigation system [49]. Further research is needed to explore the combined effects of nano-TiO_2_ with other stress mitigation measures and to assess its long-term performance and environmental safety in saline–alkali fields [50,51,52].

The potential human health risks of TiO_2_ NP accumulation in rice are a pivotal concern that must be addressed, even with their proven benefits in improving plant stress tolerance and yield. TiO_2_ NPs can be taken up by rice via roots or foliar absorption and translocated to edible grains [53], and their nanoscale size allows them to cross biological barriers, posing risks of chronic toxicity upon dietary intake—such as oxidative stress or cellular dysfunction [54]. Notably, rice grain accumulation of TiO_2_ NPs is dose-dependent [55], with excessive application beyond optimal concentrations increasing translocation to edible parts. While combining TiO_2_ NPs with biochar can reduce their bioavailability and grain accumulation [56], standardized risk assessments remain lacking. Atanda et al. [57] emphasized the urgent need for quantifying NP concentrations in grains via ICP-MS, evaluating dietary exposure risks, and establishing regulatory thresholds. In summary, the agronomic value of TiO_2_ NPs cannot outweigh food safety considerations. Future research must prioritize biosafety evaluations, and practical applications should comply with nanomaterial standards to balance stress mitigation efficacy with human health protection.

The main limitations of this study should be noted. First, we did not perform independent full physicochemical characterization of the tested nano-TiO_2_, and adopted the manufacturer-certified parameters of this commercial standard product. Second, we did not measure the actual suspended concentration and hydrated particle size of TiO_2_ in the spray suspension. The stable dose-dependent biological effects and high repeatability of biological replicates in this study have verified the stability of material properties, homogeneity of the suspension, reliability of the preparation protocol, and credibility of our experimental results. Nevertheless, independent material characterization and actual detection of suspension parameters can further improve the comprehensiveness and rigor of this work.

5. Conclusions

This study, for the first time, defines the variety-specific optimal foliar application concentrations of nano-TiO_2_ for salt-tolerant and salt-sensitive rice genotypes under salt stress, a novel quantitative basis for precise nanomaterial application in saline–alkaline rice cultivation. Foliar application of nano-TiO_2_ at 30 mg L^−1^ for JLY3261 and 45 mg L^−1^ for YXYZ substantially improved grain yield by enhancing spikelets per panicle, 1000-grain weight, and seed-setting rate. Nano-TiO_2_ alleviated salt stress through multiple synergistic pathways (Figure 9); i.e., physiologically, it improved the leaf area index, photosynthesis, aboveground biomass, antioxidant enzyme activities, and nitrogen metabolism, while reducing MDA content, whereas at the ionic and molecular levels, it enhanced K^+^/Na^+^ balance by regulating ion transport genes, and in terms of quality, it increased milling and head rice rates, improved protein content, and reduced chalkiness. Overall, nano-TiO_2_ synergistically enhanced yield and grain quality, providing a theoretical and practical basis for precise rice cultivation under saline–alkaline conditions.