Comparative analysis of chemically and green synthesized titanium dioxide nanoparticles for the regulation of photosynthesis in Lactuca sativa L.

Abstract

Green synthesized titanium dioxide nanoparticles (g-TiO₂ NPs) have aroused widespread interest in agriculture. Nevertheless, whether they are safer than chemically synthesized TiO₂ NPs (c-TiO₂ NPs) remains to be demonstrated. Herein, the photosynthetic response of Lactuca sativa L. was evaluated by foliar spraying of 10, 100, 250, and 500 mg L⁻¹ c-TiO₂ NPs or g-TiO₂ NPs. Results indicated that NPs interfered with nutrient accumulation and the cellular redox state, and all treatments displayed inhibition of growth except the 10 mg L⁻¹ g-TiO₂ NP group. Photosynthetic parameters and FTIR analysis revealed that NP stress on photosynthesis was also manifested by lower photosystem activity (500 mg L⁻¹) and disrupted Calvin cycle metabolism, but carbohydrates and proteins were more sensitive to c-TiO₂ NPs and g-TiO₂ NPs, respectively. Notably, both NPs promoted photosynthetic electron transfer (≤250 mg L⁻¹), thus alleviating detrimental effects due to suppressed ATPase and Rubisco activity, thylakoid lysis, and chloroplast autophagy. Differently, chloroplasts were more responsive to ultraviolet (non-directly utilizable) and visible light under c-TiO₂ NP treatment, which facilitated the Hill reaction while posing a photo-oxidative risk to plants. In contrast, g-TiO₂ NPs were less phytotoxic, as evidenced by higher NADPH, ATPase activity, chlorophyll, and photosynthetic efficiency, and less chloroplast damage, where 10 mg L⁻¹ g-TiO₂ NPs effectively activated the plant defense system and improved light capture and conversion. Collectively, g-TiO₂ NPs showed lower phytotoxicity by modulating energy conversion processes in chloroplasts, which provided a cutting-edge research perspective for the application of nanopesticides.

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Environmental significance

Recently, green synthetic nanotechnology with cost and environmental benefits has attracted great attention. Chemically synthesized TiO₂ nanoparticles (c-TiO₂ NPs) can stress plants when improving plant growth and the soil environment, but it is unclear whether green synthesized TiO₂ (g-TiO₂) NPs can mitigate these nanotoxicities. Herein, the effects of c-TiO₂ NPs and g-TiO₂ NPs on lettuce photosynthesis were explored by foliar spraying. The results indicated that both NPs facilitated photosynthetic electron transfer, while interference with energy synthesis and defense systems attenuated this advantage. In contrast, g-TiO₂ NPs demonstrated lower toxicity to photosynthesis by modulating light harvesting, phototransformation, and chloroplast activity. These findings revealed the potential of g-TiO₂ NPs in agricultural development and provided new insights into the risk assessment of environment-friendly nanoparticles.

1. Introduction

Nanotechnology is considered one of the most prospective technologies of the 21st century,¹ and its core building blocks, nanoparticles (NPs), are extensively applied in industrial and medical fields due to their special physicochemical and optoelectronic properties.² In recent years, NPs have also brought great promise for the development of agriculture, in particular the exploitation of nanopesticides and nanofertilizers not only reduced the overuse of traditional pesticides (20–30%)³ and the pressure on the environment, but also improved the efficiency of agricultural inputs and the nutritional value of crops (2.8–18%).⁴ It has been reported that Ag-based, Ti-based, and Cu-based nanomaterials are the commonest analyzed metal-based nanopesticides, where titanium dioxide (TiO₂) NPs with excellent photocatalytic properties, high stability, and low cost are widely used as antimicrobial agents.^4,5 Nonetheless, TiO₂ NPs are identified by the Organisation for Economic Co-operation and Development (OECD) as priority contaminants for ecotoxicity testing.⁶ Furthermore, considering the limitations of conventional pesticides, there is an urgent need to carefully review pesticides in the form of NPs to ensure their efficacy and safety.

Currently, TiO₂ NPs can be synthesized by hydrothermal, microwave irradiation, and sol–gel methods,⁷ but these methods require hazardous chemicals and extreme reaction conditions, which are time-consuming, energy-wasting, and environmentally unfriendly.⁸ Therefore, plant- and microbial-mediated synthesis of NPs is of increasing interest to researchers. Synthesis using plant extracts is generally green, safe, and cost-effective, and by-products are less toxic and easy to handle, due to the antioxidants and secondary metabolites contained in the extracts that act as bio-capping and reducing agents to stabilize the formation of NP crystals.⁹ These advantages provide a strong security guarantee for the agricultural application of NPs. It has been shown that green synthesized NPs improved plant growth by enhancing antimicrobial properties,¹⁰ protecting defense systems,¹¹ promoting nutrient uptake,¹² and mitigating heavy metal stress¹³ compared to chemically synthesized NPs. This showed that the green synthesis method is a substitute process to improve the biocompatibility of NPs and alleviate phytotoxicity, but previous studies have not systematically elucidated why the products of this process have lower phytotoxicity from the source of energy synthesis. Moreover, most data on the interaction of TiO₂ NPs with plants were based on c-TiO₂ NPs, while key evidence for the practical application of g-TiO₂ NPs in agriculture is lacking.

It is generally believed that NPs can accumulate in plant tissues through roots or leaves, but no consistent results have been obtained regarding the effects of NPs on plants. TiO₂ NPs not only improve crop growth¹⁴ and enhance stress tolerance,⁵ but also cause abnormalities in metabolism,¹⁵ gene expression,¹⁶ and mineral nutrient uptake.¹⁷ Typically, mitochondria and chloroplasts are the main organelles attacked by nanopesticides, where chloroplasts are closely related to plant growth and very sensitive to environmental pollutants as the energy synthesis factory of plants. Middepogu et al.¹⁸ found that 20 mg L⁻¹ TiO₂ NPs reduced light absorption of Chlorella pyrenoidosa through the shading effect and entered the chloroplast stroma causing damage to thylakoids. Hong et al.¹⁹ demonstrated that photosynthesis-related genes in Brassica chinensis L. were sensitive to 350 mg kg⁻¹ cerium oxide (CeO₂) NPs, ultimately manifesting in reduced carbon sequestration efficiency. Lu et al.²⁰ sprayed 20 mg mL⁻¹ iron oxide (Fe₂O₃) NPs inducing significant production of hydroxyl radicals (·OH) that decomposed chlorophyll in Triticum aestivum. Overall, chemically synthesized NPs may disrupt photosynthesis, so can NPs synthesized with bioactive components regulate the operation of energy factories to reduce harm to non-target crops?

In this study, the widely consumed and cultivated Lactuca sativa L. was identified as a model crop, and TiO₂ NPs synthesized from plant extracts and traditional chemicals were selected to (1) explore the effects of TiO₂ NPs on lettuce growth (physiological and biochemical), (2) investigate changes in photosynthetic parameters and metabolites in the light reaction and Calvin cycle, (3) elucidate the influence mechanism of TiO₂ NPs on photosynthesis in terms of energy required for photoreaction, chloroplast structure, cell management mechanism, and NP properties, and (4) compare the regulatory differences of TiO₂ NPs synthesized by distinct methods on plant growth and photosynthesis. This study provides valuable information for the efficient and safe application of TiO₂ NPs in agriculture, which will contribute to the sustainable development of nanopesticides.

2. Materials and methods

2.1 Preparation and characterization of NPs

Anatase c-TiO₂ NPs (5–10 nm, 99.8%) were purchased from Ronghua Sci. & Tech. Co. (Nanjing, China). The g-TiO₂ NPs were prepared with reference to Goutam et al.²¹ The detailed procedure is described in Text S1.† Energy dispersive spectroscopy (EDS, Octane Plus, USA) and X-ray diffraction (XRD, Ultima IV, Japan) indicated the successful synthesis of anatase g-TiO₂ NPs (Fig. S1 and S2†). Average sizes of c-TiO₂ NPs (9.3 nm) and g-TiO₂ NPs (20.1 nm) were detected by scanning electron microscopy (SEM, ZEISS Gemini 300, Germany) (Fig. S3†).

2.2 Exposure and plant growth

Due to hydroponics being clean, fertilizer-saving, and high-yield, Lactuca sativa L. has been successfully grown commercially in hydroponic systems, the more easily controlled hydroponics was chosen as the culture method to simulate actual exposure scenarios. Lettuce seeds were provided by Hezhiyuan Seed Corporation (Shandong, China). The seeds were disinfected with 1% NaClO and grew for 3 weeks in a tray containing ⅛-strength Hoagland nutrient solution, and then the seedlings were transferred to triangle bottles containing ¼-strength Hoagland nutrient solution for 1 week. 10, 100, 250, and 500 mg L⁻¹ NPs were uniformly sprayed on lettuce leaves with small sprayers from 9 a.m. to 10 a.m. for 14 consecutive days from the 5th week (2.5 ml per day per plant), and the TiO₂ NP suspension was ultrasonicated for 30 min before exposure. Control checks (CK) were performed with water instead of NPs. At the same time, the bottle was covered to prevent the hydroponic substrate from being contaminated. Each group was supplemented with an equal amount of nutrient solution every 2 days. There were three replicates per treatment and all incubations were performed in a light incubator with a diurnal cycle of 16/8 h (23/18 °C).

2.3 Physiological and photosynthetic index testing

2.3.1 Growth parameters and nutrient content. Fresh weights of lettuce tissues treated for 2 weeks were determined after ultrasonic cleaning with EDTA-Na₂, and dry weights were obtained after drying. The dried leaves were digested with concentrated H₂SO₄ and H₂O₂ (2 : 1) at 200 °C until the solution was clear and then it was filtered to obtain the digestion solution. Elemental contents were detected by atomic absorption spectroscopy (AAS, PerkinElmer Optima 8000, USA).

2.3.2 Measurement of oxidative stress indicators. Fresh leaves were placed in a pre-chilled mortar and homogenized with 0.05 M 7.8 pH phosphate buffer solution (PBS) at a ratio of 1 : 10 (w : v) in an ice-cold environment, and the mixture was centrifuged to obtain a crude enzyme extract. The activities of catalase (CAT) and ascorbate peroxidase (APX) were determined as explained by Kohatsu et al.¹¹ The superoxide dismutase (SOD) activity was measured following the assay published by Ji et al.²² The reduced glutathione (GSH) content was analyzed according to Huang et al.²³

2.3.3 Photosynthetic parameter analysis. The net CO₂ assimilation rate (A), stomatal conductance (G_s), intercellular CO₂ concentration (C_i), and transpiration rate (E) were determined with a portable photosynthesis measurement system (TARGAS-1, Hansatech, UK). A pulse-modulated fluorometer (FMS 2, Hansatech, UK) was used to calculate the maximum quantum efficiency (F_v/F_m), photochemical efficiency of photosystem II (ΦPSII), photochemical quenching (qP), non-photochemical quenching (NPQ) and photosynthetic electron transport rate (ETR). The chlorophyll content was measured using the ethanol method.

2.3.4 Assessment of changes in biomolecules. Fourier Transform Infrared (FTIR, Spotlight 200i, PerkinElmer) spectroscopy was adopted to obtain changes in leaf biomolecules. Band assignments based on previous studies are given in Table S1.† Two-dimensional correlation spectroscopy (2D-COS) was utilized to improve the resolution of the spectra, and the amide I region was further analyzed using the second derivative spectra. The average results from three replications were analyzed and plotted. See Text S2† for a detailed description.

2.4 Determination of energy and metabolites in photosynthesis

The contents of nicotinamide adenine dinucleotide phosphate (NADPH) and protein in leaves were extracted and determined using reagent kits (Nanjing Jiancheng Biotechnology Institute, China). The activities of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) and adenosine triphosphate synthase (ATPase) in leaves were measured using an enzyme-linked immunosorbent assay (ELISA) kit (Beijing Libo Biotechnology Co., Ltd., China). The soluble sugar content in leaves was assayed with anthrone colorimetry.

2.5 Hill reaction and optical property monitoring

Chloroplast suspensions were extracted as defined by Pan et al.²⁴ and the chlorophyll concentration was 360 mg L⁻¹. Chloroplast suspensions were mixed with 10, 100, 250, and 500 mg L⁻¹ TiO₂ NPs (1 : 1), respectively, and incubated in the dark for 2 h to obtain TiO₂ and chloroplast complexes (TiO₂/Chl). The Hill reaction was performed to evaluate the effects of TiO₂ NPs on photosynthetic activity. The reduction of 2,6-dichlorophenol indophenol (DCPIP) and potassium ferricyanide was determined with reference to Li et al.²⁵ with minor modifications. The difference in photoelectron generation rates of c-TiO₂ NPs and g-TiO₂ NPs was identified by methylene blue (MB) reduction. UV-vis spectra of TiO₂/Chl were determined on a spectrophotometer (Spotlight 200i, Perkin Elmer). Refer to Text S3† for the detailed process.

2.6 Observation of subcellular structure

After 14 days of exposure, leaves from the control, 10 mg L⁻¹ g-TiO₂ NP, 500 mg L⁻¹ g-TiO₂ NP, and c-TiO₂ NP treatments were selected for glutaraldehyde fixation. After rinsing with 0.1 M PBS, the leaves were immersed in 1% osmium acid solution, dehydrated in gradient ethanol, and transferred to acetone solution. Subsequently, the tissues were put in a mixture of embedding agent and acetone and then left overnight in the pure embedding agent. Finally, sections made with an ultramicrotome (Leica EM UC7, Germany) were stained with lead citrate and uranyl acetate, and chloroplast structures were examined using a transmission electron microscope (TEM, Hitachi H-7650, Japan).

2.7 Visualization of autophagosome

To visualize the structure of intracellular autophagy, monodansylcadaverine (MDC) staining was performed following the method by Wang et al.²⁶ with minor modifications (Text S4†). Briefly, protease inhibitor-treated leaves were immediately vacuum stained with 100 μM MDC (Sigma-Aldrich) in the dark for 30 min at a constant temperature, then rinsed with pH 7.4 PBS and observed under confocal laser scanning microscopy (CLSM, LSM 800, Zeiss, Germany) for fluorescence from MDC and chloroplasts.

2.8 Statistical analysis

All data in this work were displayed as mean ± standard deviation (SD). One-way ANOVA was performed using IBM SPSS Statistics 26 and combined with Duncan's multiple comparison test, and the significance level of all results was defined as p < 0.05.

3. Results and discussion

3.1 Disturbance of antioxidant defense system and growth

The effects of TiO₂ NPs on lettuce growth were assessed by measuring changes in biomass and inorganic nutrients. As seen in Fig. 1, both NPs negatively affected growth at concentrations above 100 mg L⁻¹, and c-TiO₂ NPs exhibited greater phytotoxicity. It is worth noting that 10 mg L⁻¹ g-TiO₂ NPs increased the fresh weight of roots and leaves by 1.35 and 1.17 times over the control (Fig. 2A). Furthermore, TiO₂ NPs interfered with macro- and micro-nutrient uptake. Specifically, most plants contained fewer elements after exposure, but 10 mg L⁻¹ c-TiO₂ NPs (Mn, Zn) and g-TiO₂ NPs (Ca, Mg, and Mn) promoted elemental absorption (Fig. 2C). These elements are closely related to plant development, and an imbalanced intake of any of them can lead to physiological disorders. Xie et al.²⁷ found that CeO₂ NPs may affect mineral accumulation in Phaseolus vulgaris by interfering with ion transport channels, thus directly limiting plant growth. Consequently, the reduction of nutrients caused by NPs is one of the causes of lettuce biomass decrease.

Fig. 1 Morphology of lettuce leaves and roots in different treatment groups and indications for phytotoxic intensity of c-TiO₂ NPs and g-TiO₂ NPs. Numbers denote the proportion of up-regulation (blue) or down-regulation (red) in leaf fresh weight at 500 mg L⁻¹ compared to the control, with darker colors meaning greater intensity.

Fig. 2 Fresh weight and dry weight of leaves and roots (A), antioxidant enzyme activities in leaves (B), and changes of inorganic elements in leaves (C), values in cells represent fold change in content compared to the control, red indicates increase and blue reflects decrease. Different letters represent significant differences (p < 0.05). c-: c-TiO₂ NPs and g-: g-TiO₂ NPs.

As a by-product of aerobic metabolism, low levels of reactive oxygen species (ROS) are involved in regulating plant physiological activities, but external stresses can break the balance between ROS production and clearance, at which point plants produce enzymatic and non-enzymatic antioxidants to maintain cellular stability. As shown in Fig. 2B, TiO₂ NPs disrupted the redox metabolic status of lettuce. Compared with the control, 10 mg L⁻¹ g-TiO₂ NPs significantly increased SOD, CAT, and APX activity. The SOD activity showed a U-shaped curve trend and was strongly inhibited at 500 mg L⁻¹, but was unrelated to the synthesis method. SOD is often considered the first line of defense against oxidative stress by disproportionating superoxide radicals (·O₂⁻) to H₂O₂ and O₂,²⁸ after which APX is involved in the removal of H₂O₂, and the remaining H₂O₂ can be reduced to H₂O by CAT.²⁹ Thus, the diminished APX activity in all treatment groups except 10 mg L⁻¹ g-TiO₂ NPs indicated that APX could not completely decompose H₂O₂, which further stimulated CAT (Fig. 2B). The increased antioxidant enzyme activity was a compensatory mechanism for ROS accumulation in lettuce, but excess ROS inhibited activity and may even oxidatively damage proteins, lipids, and DNA.²⁷ Moreover, except for enzymatic reactions, which are the preferred pathway for ROS detoxification, non-enzymatic antioxidants play an invaluable role in resisting oxidative stress. In Fig. S4,† the GSH content decreased dramatically only at 500 mg L⁻¹ c-TiO₂ NPs, which was caused by the oxidation of GSH to oxidized glutathione (GSSG) by uncleared ROS. The above findings revealed that c-TiO₂ NPs and g-TiO₂ NPs activated the antioxidant system, while 10 mg L⁻¹ g-TiO₂ NPs had stronger oxidative protection.

3.2 Response of photosynthetic parameters

Changes in growth are directly related to photosynthesis. As observed in Fig. 3A, A was significantly enhanced after spraying of 100 mg L⁻¹ c-TiO₂ NPs (∼12%) and 10 mg L⁻¹ g-TiO₂ NPs (∼48%), so appropriate doses of TiO₂ NPs promoted photosynthesis and lettuce could synthesize more organic matter. c-TiO₂ NPs (100, 500 mg L⁻¹) and g-TiO₂ NPs (10, 500 mg L⁻¹) increased G_s and E. Theoretically, G_s was negatively correlated with C_i, but 500 mg L⁻¹ NP groups had higher C_i, indicating that CO₂ fixation was limited. Chlorophyll is an essential pigment for photosynthesis, which converts light energy into chemical energy. In Fig. S5,† the content of chlorophyll presented dose-dependent decreases with increasing exposure time and g-TiO₂ NPs were less toxic. In contrast, 10 mg L⁻¹ g-TiO₂ NPs increased photosynthetic pigments. This difference can be attributed to the effect of TiO₂ NPs on the uptake of minerals (Mg, Mn, etc.) (Fig. 2C), as Mg is important for chlorophyll synthesis, and Fe, Mn, and Zn are auxiliary elements during photosynthetic pigment formation.^30,31 On the other hand, ·OH, which cannot be scavenged by the plant antioxidant system, is a very strong non-selective oxidant that can decompose chlorophyll. These results showed that TiO₂ NPs inhibited light absorption and conversion by impeding chlorophyll biosynthesis or promoting chlorophyll degradation, while 10 mg L⁻¹ g-TiO₂ NPs resulted in more Mg accumulation and minimized ROS impairment, thereby mitigating phytotoxicity.

Fig. 3 Gas exchange parameters of lettuce (A) and chlorophyll fluorescence parameters (B and C). c-: c-TiO₂ NPs, g-: g-TiO₂ NPs.

Photosynthetic fluorescence parameters were measured to gain more information about the influence of TiO₂ NPs on the photosystem. As shown in Fig. 3B and C, g-TiO₂ NPs had higher ΦPSII, ETR, and qP than c-TiO₂ NPs and exhibited the greatest positive effect on ΦPSII or ETR at 10 and 250 mg L⁻¹, respectively, but both were lower than the control at the highest concentration. It was found that all qP parameters were above the untreated group, indicating that both NPs enhanced the photochemical reaction of lettuce. The impeded ETR may be owing to the lack of Fe and Cu in the leaves (Fig. 2C) because the electron transport from Q_A to Q_B requires Fe catalysis,³² and the electron transfer from the cytochrome b₆f complex to photosystem I (PSI) is associated with Cu.³³ More information on the correlation between TiO₂ NPs and electron transfer will be further explored below. In addition, insufficient levels of Mn, an important metal cofactor for electron transfer in PSII, were also associated with decreased ΦPSII and ETR. F_v/F_m reflects the photosynthetic efficiency of PSII under dark conditions, i.e., the number of absorbed photons that can drive electron transport.³⁴ In all experimental groups, the F_v/F_m values of lettuce exposed only to 100 mg L⁻¹ c-TiO₂ NPs and 10 mg L⁻¹ g-TiO₂ NPs were higher than the control (Fig. 3B and C). The inhibition of F_v/F_m confirmed that the PSII reaction center was disrupted. NPQ prevents photodamage by dissipating excess excitation energy and is an embodiment of plant photoprotective capacity.³⁵ It can be observed that NPQ under 10 mg L⁻¹ g-TiO₂ NP treatment was only 0.88 of the control, suggesting that lettuce could use light energy more efficiently and reduce heat dissipation. Other groups of g-TiO₂ applications had higher NPQ than c-TiO₂, which might be due to enhanced light capture by g-TiO₂ NPs. In general, g-TiO₂ NPs showed more potential in improving photoreaction efficiency, but the problem of photodamage caused by excess light energy should be noted.

3.3 Alterations in Calvin cycle metabolites

The perturbed light reaction would affect CO₂ fixation. In this work, FTIR spectroscopy was used to identify changes in lipids (2500–3500 cm⁻¹), proteins (1200–1800 cm⁻¹), and carbohydrates (800–1200 cm⁻¹) of leaves. Signals in the 3585–3100 cm⁻¹ band identified in the control were weakened in the treated samples (Fig. 4A). Notably, new peaks appeared especially under g-TiO₂ NP treatment at 1551 cm⁻¹ and 1460 cm⁻¹ in the amide II region. These findings implied that g-TiO₂ NPs have the potential to increase the amide species of protein. The higher dose of NPs also attenuated the peak intensity corresponding to amide III (1240 cm⁻¹). Besides, NPs adversely impacted carbohydrates, as exemplified by the weakened peak strength at 1052 cm⁻¹ and 1161 cm⁻¹ representing cellulose, and the peak at 824 cm⁻¹ associated with sugar skeleton vibration almost disappeared. Similar to our findings, Bakshi et al.³⁶ found that TiO₂ NPs elicited the reprogramming of macromolecular metabolism in Solanum lycopersicum.

Fig. 4 FTIR spectrum of lettuce leaves (A) and synchronous (B and C) and asynchronous (D and E) 2D-FTIR-COS maps generated from the 1800–800 cm⁻¹ region under c-TiO₂ NP (B and D) and g-TiO₂ NP (C and D) treatments. c-: c-TiO₂ NPs, g-: g-TiO₂ NPs.

It is well-known that the 1600–1700 cm⁻¹ region is characteristic of amide I,³⁷ which was observed to be deformed and attenuated. It reflects the protein secondary structure, including β-sheet (1610–1640 cm⁻¹), random coil (1640–1650 cm⁻¹), α-helix (1650–1658 cm⁻¹), and β-turn (1660–1700 cm⁻¹).³⁸ This work enhanced the identification of raw characteristic peaks by FTIR curve fitting based on the second derivative spectra to analyze protein structures (Fig. S6†). It can be seen from Table S2† that the protein structure in leaves changed from α-helix to β-sheet at 100 and 250 mg L⁻¹, and the c-TiO₂ NP treatment showed a stronger response. Normally, the transition from α-helix to β-sheet is considered a form to resist oxidative stress in adversity, such as antibiotic,³⁹ perfluorooctanoic acid,⁴⁰ perfluorooctane sulfonic acid stress,⁴¹etc. The α-helix is more compact to enhance protein tolerance of external disturbances, while the loose β-sheet provides more redox active sites, which can lead to oxidative damage of protein. From this, the increase in β-sheet conformation induced by TiO₂ NPs may cause more serious physiological or metabolic abnormalities.

To further investigate the discrepancies between the two NPs on biomolecules, concentration-dependent 2D-FTIR-COS analysis was adopted (Fig. 4B–E). The diagonal auto-peak in the 2D-FTIR-COS synchronous map means sensitivity to perturbations, while the cross-peak represents changes in the corresponding peaks that have the same mechanism and origin.⁴² In this study, most auto-peaks appeared in the 800–1800 cm⁻¹ region (Fig. S7†). Differently, the most obvious auto-peaks appeared in dissimilar regions in the c-TiO₂ NPs (800–1200 cm⁻¹) and g-TiO₂ NPs (1200–1800 cm⁻¹) spectra, signifying that carbohydrates and proteins were more susceptible to c-TiO₂ NP and g-TiO₂ NP interference, respectively. The soluble protein and starch content in leaves exhibited a similar trend (Fig. S8†). This contradicted the results of previous protein structural analysis, which may be caused by stronger changes of amide II and amide III than those of amide I. All cross-peaks with the positive signal implied that changes in biochemical components corresponding to IR signals in leaves were synchronized.

In addition, the order of change in functional groups in response to external disturbances can be further determined by comparing cross-peak signals in synchronous and asynchronous maps.⁴² The information for each cross-peak symbol is collated in Table S3† according to Noda's rule.⁴³ The functional group responded to c-TiO₂ NPs in the order 1052 cm⁻¹ > 3307 cm⁻¹ > 1243 cm⁻¹ > 1385 cm⁻¹ > 1596 cm⁻¹. Therefore, c-TiO₂ NPs first elicited disruption of carbohydrates such as cellulose, after which amides and suberin were destroyed. Likewise, leaves exposed to g-TiO₂ NPs exhibited a reaction sequence of 654 cm⁻¹ > 1385 cm⁻¹ > 1596 cm⁻¹ > 3307 cm⁻¹ > 1052 cm⁻¹. Unlike c-TiO₂ NPs, g-TiO₂ NPs elicited a more rapid change in proteins. This coincided with the sensitivity of macromolecules to NPs in synchronous maps. In conclusion, foliar application of TiO₂ NPs altered Calvin cycle metabolites in lettuce and different macromolecules presented divergent sensitivities to the two NPs. The strong interference of c-TiO₂ NPs with carbohydrates can directly cause low yields. Ahmed et al.⁴⁴ pointed out that disturbance of protein by contaminants could affect the lipid and carbohydrate content and category, so a high response of protein to g-TiO₂ NPs would pose a further threat to growth.

3.4 Mechanism of photosynthetic response induced by NPs

3.4.1 Modulation of light absorption and conversion. To explore the mechanism of TiO₂ NPs affecting the lettuce photoreaction and the Calvin cycle, important enzyme activities and the product content during photosynthesis were measured. It is widely known that electrons in photosystems can be transferred to NADP⁺ to reduce it to NADPH. The NADPH yield can reflect the rate of photosynthetic electron transport.⁴⁵ As shown in Table 1, NADPH was more influenced by g-TiO₂ NPs, which were all more than twice as much as the control, suggesting that g-TiO₂ NPs increased the number of electrons in the PSI. Meanwhile c-TiO₂ NPs increased NADPH by 42% only at 250 mg L⁻¹. Thereby, the discrepancy between the two NPs on photosynthetic electron transport should not be ignored. ATPases play a critical role in the phosphorylation process of photosynthesis. The NPs inhibited ATPase activity except for 10 mg L⁻¹ g-TiO₂ NPs (up 14%), which directly down-regulated ATP production in the photochemical phase. Moreover, insufficient NADPH and ATP will prevent the process of carboxylation, reduction, and regeneration in the Calvin cycle,³³ because 3-phosphoglycerate (PGA) and glyceraldehyde-3-phosphate (GAP) as photosynthetic intermediates cannot be converted without catalysis of ATP and NADPH. Carboxylation is the first step for CO₂ to enter the plant, and this process is regulated by Rubisco, which catalyzes CO₂ into energy-rich metabolites.⁴⁶ The Rubisco activity decreased after the application of NPs, where it was significantly higher under g-TiO₂ NP treatment than c-TiO₂ NP treatment at 10 and 100 mg L⁻¹, but the opposite was true at 500 mg L⁻¹, which may be related to the greater sensitivity of protein to g-TiO₂ NPs (Fig. 4C). Moreover, the ROS burst also reduced the Rubisco activity.⁴⁷ The CO₂ fixation efficiency of Rubisco is only 1–10 s⁻¹,⁴⁸ making it a rate-limiting enzyme for photosynthesis, so the decrease in its activity inhibited carbohydrate synthesis and thus affected the respiratory process.⁴⁷ Middepogu et al.¹⁸ confirmed that restriction of Chlorella pyrenoidosa development by TiO₂ NPs stemmed from inhibition of ATPase activity. Therefore, even though TiO₂ NPs reduced more NADP⁺ by facilitating photosynthetic electron transfer, their inhibition of ATPase and Rubisco resulted in the underutilization of NADPH, thereby limiting the conversion of PGA and GAP to downstream metabolites such as amino acids (Fig. S8A†), sugars (Fig. S8B†), and fatty acids.

Table 1 Influences of TiO₂ NPs on photosynthesis energy and the ability of TiO₂ NPs or TiO₂/Chl to generate electrons

Treatment	Dose (mg L^−1)	NADPH content (nmol g^−1 FW)	ATPase activity (U mg^−1 prot)	Rubisco activity (U g^−1 FW)	Reduction (%)
					DCPIP	Ferricyanide	MB
Note: different letters represent significant differences (p < 0.05).
Control	0	6.70 ± 0.10^e	3.46 ± 0.10^b	0.29 ± 0.004^a	19.21 ± 0.15^e	39.47 ± 1.49^e	13.75
c-TiO2 NPs	10	5.69 ± 0.20^e	3.13 ± 0.09^c	0.27 ± 0.004^c	23.28 ± 0.78^de	43.81 ± 1.51^de	26.41
	100	6.12 ± 0.10^e	2.08 ± 0.08^ef	0.24 ± 0.005^ef	47.85 ± 0.73^c	58.41 ± 0.93^c	35.66
	250	9.50 ± 0.20^d	1.89 ± 0.12^f	0.25 ± 0.003^e	54.46 ± 1.26^b	71.65 ± 1.91^b	39.74
	500	5.47 ± 0.61^e	2.89 ± 0.11^cd	0.24 ± 0.003^f	65.69 ± 1.56^a	75.47 ± 1.36^ab	70.04
g-TiO2 NPs	10	20.45 ± 0.41^a	3.94 ± 0.15^a	0.28 ± 0.002^b	28.76 ± 0.27^d	47.92 ± 1.75^d	30.07
	100	14.54 ± 0.20^c	2.64 ± 0.18^d	0.26 ± 0.004^d	42.22 ± 1.28^c	56.77 ± 1.92^c	34.39
	250	17.14 ± 1.02^b	2.27 ± 0.10^ef	0.24 ± 0.002^f	58.10 ± 0.46^b	75.68 ± 1.78^ab	50.12
	500	14.26 ± 0.81^c	2.93 ± 0.11^c	0.23 ± 0.003^g	67.43 ± 3.85^a	78.73 ± 1.77^a	75.27

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It has been discussed in section 3.2 that the decrease in ETR was due to the deficiency of crucial elements in the photosynthetic electron transfer process; meanwhile, could the increased ETR under NP treatment (≤250 mg L⁻¹) be related to the photoelectric properties of TiO₂ NPs? To address this issue, MB degradation by TiO₂ under light was measured, and the interaction between NPs and plants at the organelle level was explored using chloroplasts isolated from lettuce. As seen in Table 1, MB degradation increased with dose after irradiation with xenon lamps for 3 h, which was accompanied by photogenerated electrons from NPs, while g-TiO₂ NPs showed higher effects in most groups. Subsequently, the Hill reaction was used to verify that this light-absorbing capacity of NPs could be utilized by chloroplasts to increase photosynthetic activity. DCPIP was chosen as the electron acceptor to intercept electrons in the photosystem.⁴⁹ The reduction of DCPIP was faster in treated groups than in chloroplasts alone, but this was independent of the synthesis method. Then, the ferricyanide reduction experiment by TiO₂/Chl was adopted to further determine whether enhanced Hill activity was related to NPs, whose reaction trend was similar to DCPIP, and there were no significant differences between the two NPs. The above results indicated that TiO₂ NP application supplemented the electrons in photosystems, thus improving the photosynthetic efficiency. In Fig. S9,† chloroplasts showed two absorption bands at 400–500 nm and 650–700 nm and TiO₂/Chl complexes had the same absorption pattern as bare chloroplasts, but with greater absorption intensity, in which c-TiO₂/Chl exhibited more pronounced absorption. Combined with Table S4,† g-TiO₂ NPs had stronger adsorption capacity because they were more dispersed in the aqueous solution, which explained why g-TiO₂ NPs with weak UV absorption degraded MB faster instead.

Therefore, the greater advantage of g-TiO₂ NPs over c-TiO₂ NPs in enhancing NADPH and ETR is mainly due to improved electron transport processes, such as elemental uptake, chloroplast activity, and cellular homeostasis, rather than the effect of the optoelectronic properties of NPs. Notably, the enhanced absorption of the UV-A region stressed plants after exposure to NPs (Fig. S9†),⁵⁰ while excess electrons in the light reaction triggered a ROS burst that inhibited photosynthesis,²⁵ which explained the decrease in antioxidant enzyme activity at high concentrations as shown in Fig. 2B. Therefore, even though both TiO₂ NPs increased electrons in the photosynthetic chain, their inhibition of critical enzyme activities and induction of ROS counteracted some positive effects.

3.4.2 Disruption of the cellular ultrastructure. In addition to the limitation of photosynthetic reactions, metabolic disorders stemming from the disrupted chloroplast structure may be another critical reason for poor photosynthetic efficiency. Chloroplasts showed diverse structural features as observed from the TEM images (Fig. 5). Chloroplasts in the control group were clear in the cytoplasm and were ellipse-aligned along the cell wall, indicating vacuole integrity and a normal chloroplast shape. Also, there were more chloroplasts and starch grains in the control, and the thylakoid layer was clearly distributed and tightly clustered (Fig. 5A–C). The difference was that thylakoids tended to loosen and dissociate after 10 mg L⁻¹ g-TiO₂ NP treatment (Fig. 5D–F). Under 500 mg L⁻¹ treatment, the number of chloroplasts and starch grains decreased, and osmiophilic granules increased, where c-TiO₂ NPs reduced thylakoid stacking (Fig. 5G–J).

Fig. 5 Ultrastructure of lettuce chloroplasts under control (A–C), g-10 (D–F), g-500 (G–I), and c-500 (J–L) treatments. CW: cell wall, Ch: chloroplast, S: starch granule, CM: cell membrane, T: thylakoid, O: osmiophilic granule, N: nucleus, V: vacuole, c-: c-TiO₂ NPs, g-: g-TiO₂ NPs. Arrows indicate autophagosomal-like structures.

Normally, thylakoids are stacked into grana in the chloroplast,¹⁸ so the dissolution of their lamellar structures was an important reason for blocked light absorption. Besides, the increased osmiophilic granules suggested that TiO₂ NPs elicited oxidative damage in plants, as they are usually a symbol of oxidative stress,⁵¹ which was strongly supported by the significant inhibition of enzymatic and non-enzymatic antioxidants at 500 mg L⁻¹. The decrease in starch granules was the result of their degradation or reduced efficiency of carbon assimilation. It was noted that starch degradants are osmotic and can lead to inflation of organelles,¹⁷ which explained the deformation and over-swelling of chloroplasts observed in Fig. 5D, G and J. Starch drives most carbon fluxes in plants and occupies an essential position in carbon allocation, and its deficiency can directly restrict crop growth (Fig. S8B†). Previous studies have confirmed that NPs can be internalized by leaves through the cuticle (0.6–4.8 nm)⁵² and stomata (about 20–40 μm long and 5–10 μm wide).⁵³ Therefore, smaller c-TiO₂ NPs were more likely to enter cells through the stomata than g-TiO₂ NPs in this work, thus posing a greater threat to the integrity of chloroplasts and thylakoid membranes. In summary, TiO₂ NPs had negative effects on thylakoids even at low concentrations, and higher doses induced chloroplast degradation, but this phenomenon was more pronounced under c-TiO₂ NP exposure.

3.4.3 Occurrence of autophagic response. It has been confirmed that autophagy is a mechanism for modulating cellular constituents. When faced with abiotic stresses including heat, high salinity, and drought, plants remove unwanted intracellular components by triggering an autophagic signaling cascade in vivo. Alternatively, autophagy can also regulate critical metabolic processes by decomposing certain organelles or cells during normal growth.⁵⁴ During autophagy, the sequestering membrane called the phagophore or isolation membrane will engulf the cytoplasm and eventually form an autophagosome with a double-membrane structure.⁵⁵ According to the autophagosomal-like structures in Fig. 5G and K, it was speculated that TiO₂ NPs might trigger the intracellular autophagic response. To further verify the conjecture, MDC, which specifically stains autophagosomes, was selected to label acidic spaces. There were no obvious punctate fluorescence structures in leaves treated without NPs and 10 mg L⁻¹ g-TiO₂ NPs (Fig. 6A). However, the number of MDC-labeled autophagosomes increased appreciably at high concentrations, and numerous fluorescent spots were observed around chloroplasts, especially after c-TiO₂ NP coercion (Fig. 6C and D). This was consistent with the quantitative analysis of MDC fluorescence, where the MDC fluorescence intensity under 500 mg L⁻¹ g-TiO₂ NP and c-TiO₂ NP treatments was 3.2-fold and 8.6-fold stronger than the control, respectively (Fig. 6B). Higher than normal levels of autophagy showed that more organelles were destroyed by TiO₂ NPs, which was a manifestation of nanotoxicity.

Fig. 6 CLSM images of leaf tissue stained by MDC (A), the relative fluorescence intensity of MDC (B), and enlarged views of blue dashed boxes in g-500 (C) and c-500 (D) groups in A, respectively. MDC-labeled structures are blue and chloroplasts are red. Quantification of autophagic activity relative to the control by calculating the mean fluorescence intensity in 6 fields of view. Different letters represent significant differences (p < 0.05). c-: c-TiO₂ NPs, g-: g-TiO₂ NPs.

The prerequisites for autophagy are usually inextricably linked to a burst of ROS, which oxidizes cellular macromolecules and then leads to organelle damage and cytological dysfunction. Consequently, as a plant housekeeping mechanism, autophagy will clean up these components destroyed by excess ROS induced by TiO₂ NPs. Colocalization of chloroplast and MDC fluorescence suggested that TiO₂ NPs activated the autophagic degradation of chloroplasts. This was proved in Fig. S10,† where 500 mg L⁻¹ TiO₂ NPs reduced the number of chloroplasts and their fluorescence intensity, but inhibition was greater under c-TiO₂ NP treatment. Summarizing the above results, the photosynthetic response model of lettuce to TiO₂ NPs is illustrated in Scheme 1. Additionally, Izumi et al.⁵⁵ reported that the breakdown of Rubisco was closely related to autophagy in rice. Since approximately half of the soluble proteins in leaves are derived from Rubisco, the decrease in Rubisco activity (Table 1) and soluble protein content (Fig. S8A†) was also associated with chloroplast autophagy. On the other hand, plants can also maintain survival under stress by limiting programmed cell death⁵⁶ and recycling beneficial components⁵⁷ through autophagy. Collectively, higher levels of TiO₂ NPs triggered the autophagic protection mechanism in lettuce but reduced the activity and number of chloroplasts, which was detrimental to the utilization of light energy.

Scheme 1 Schematic representation of TiO₂ NPs modulating photosynthesis in lettuce. The left rectangle of the color bar represents c-TiO₂ NPs, and the right means g-TiO₂ NPs. Red indicates up-regulation, blue indicates down-regulation, and the shade of color reflects the intensity of regulation. Fd, ferredoxin; Cytb₆f, cytochrome b₆f complex; PQ, plastoquinone; PQH₂, plastoquinol; PC, plastocyanin; RuBP, ribulose 1,5-bisphosphate; GAP, glyceraldehyde-3-phosphate; PGA, 3-phosphoglycerate.

4. Conclusion

This study evaluated the effects of lettuce photosynthesis by foliar spraying of TiO₂ NPs synthesized with different methods and investigated the modulatory principles of TiO₂ NPs on photosynthesis in terms of light energy conversion, chloroplast structure, cellular management mechanisms, and NP properties. Results revealed that c-TiO₂ NPs and g-TiO₂ NPs exert dose-dependent impacts on plant growth. They perturbed the accumulation of nutrients, including Mg, Fe, Cu, Zn, and Mn, thereby impeding chlorophyll synthesis and photosynthetic electron transport. NPs promoted chloroplast absorption of light at 300–800 nm wavelength, but this advantage caused a burst of oxidative radicals at high concentrations, which induced thylakoid lysis, chlorophyll degradation, and defense system destruction, and eventually evoked a decline in photosynthetic efficiency and resulted in disruption of carbon metabolism. Therein, these phenomena were more obvious when exposed to c-TiO₂ NPs with superior photoelectric properties. Furthermore, direct biomass loss in leaves (2–35%) and roots (23–44%) resulted from a stronger response of carbohydrates to c-TiO₂ NPs, while high susceptibility of proteins to g-TiO₂ NPs posed a threat to carbohydrates and lipids during long-term exposure. Notably, 10 mg L⁻¹ g-TiO₂ NPs alleviated stress by regulating pigment synthesis, photophosphorylation, antioxidant enzymes, and chloroplast activities. Overall, g-TiO₂ NPs show greater potential for agricultural development than c-TiO₂ NPs and play a critical role in modulating plant photosynthesis when used appropriately, providing additional evidence for the efficient combination of nanotechnology and plant protection products. It should be noted that foliar exposure to NPs altered nutrient uptake, so further verification of root damage is necessary, and health risks of titanium levels in plants also need to be considered before practical application.

Author contributions

Yuzhu Weng: conceptualization, methodology, investigation, formal analysis, writing – original draft, writing – review & editing; Xue Bai: conceptualization, project administration, funding acquisition, supervision, writing-review & editing; Mengen Kang: investigation, writing – review & editing; Yue Huang: methodology, investigation; Yetong Ji: writing – review & editing; Haoke Wang: methodology; Zulin Hua: funding acquisition.

Conflicts of interest

There are no conflicts of interest to be declared.

Acknowledgements

The authors gratefully acknowledge the support provided by the National Natural Science Foundation of China (U2243601) and the Priority Academic Program Development of Jiangsu Higher Education Institutions of China.

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Footnote

† Electronic supplementary information (ESI) available. See DOI: https://doi.org/10.1039/d3en00589e

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