Plants exposed to titanium dioxide nanoparticles acquired contrasting photosynthetic and morphological strategies depending on the growing light intensity: a case study in radish

Abstract

Due to the photocatalytic property of titanium dioxide (TiO₂), its application may be dependent on the growing light environment. In this study, radish plants were cultivated under four light intensities (75, 150, 300, and 600 μmol m⁻² s⁻¹ photosynthetic photon flux density, PPFD), and were weekly sprayed (three times in total) with TiO₂ nanoparticles at different concentrations (0, 50, and 100 μmol L⁻¹). Based on the obtained results, plants used two contrasting strategies depending on the growing PPFD. In the first strategy, as a result of exposure to high PPFD, plants limited their leaf area and send the biomass towards the underground parts to limit light-absorbing surface area, which was confirmed by thicker leaves (lower specific leaf area). TiO₂ further improved the allocation of biomass to the underground parts when plants were exposed to higher PPFDs. In the second strategy, plants dissipated the absorbed light energy into the heat (NPQ) to protect the photosynthetic apparatus from high energy input due to carbohydrate and carotenoid accumulation as a result of exposure to higher PPFDs or TiO₂ concentrations. TiO₂ nanoparticle application up-regulated photosynthetic functionality under low, while down-regulated it under high PPFD. The best light use efficiency was noted at 300 m⁻² s⁻¹ PPFD, while TiO₂ nanoparticle spray stimulated light use efficiency at 75 m⁻² s⁻¹ PPFD. In conclusion, TiO₂ nanoparticle spray promotes plant growth and productivity, and this response is magnified as cultivation light intensity becomes limited.

Introduction

Global food demand is steadily rising as a result of a growing population¹. To ensure global food security, further improvements in crop yields are necessary^1,2. Intensifying agricultural production by increasing the associated inputs is commonly employed as an effective strategy to stimulate plant growth and performance^3–6. Yet, improving input use efficiency stands out as a more attractive alternative⁶, since the intensification-associated environmental pressure is evaded⁷.

Noteworthy, the application of nanoparticles has proven to be a powerful tool in advancing yield⁸. For instance, the application of titanium dioxide (TiO₂) nanoparticles has been associated with stimulation of carbohydrate production, chlorophyll formation, as well as improvements in photosynthetic rate and eventually yield^9,10. These promotive effects are concentration dependent, and vary excessively among species^9,11. In addition, the positive effect of TiO₂ nanoparticles on plant growth and productivity depends on the environmental conditions during cultivation^10,11. In this regard, their application may be exercised when the remaining conditions allow optimal impact on plants. For instance, plant growth and productivity also strongly depends on light availability^12–15. Light is the main energy source-driver of photosynthesis. Light intensity is the pivotal determinant of plant growth and performance^16–18. Production of crops in greenhouses and controlled environments has nowadays attracted a lot of attention¹⁹. There is a need to optimize the photosynthetic photon flux density (PPFD) needed for plant growth in an approach with the highest resource use efficiency^6,18. TiO₂ nanoparticles are used to improvemeyield during winter cultivation of plants²⁰. However, the effect of either TiO₂ nanoparticles application or light level has been limitedly addressed. It, therefore, remains unknown whether or not these two factors interact to determine plant yield.

Chlorophyll fluorescence is often employed for non-invasive assessments of the electron transport system properties^21–24. Polyphasic chlorophyll fluorescence induction curves may be additionally utilized to study the fate of absorbed light energy and provide further insights into the structure and function of the photosynthetic system^25–27.

TiO₂ nanoparticles have special properties such as high specific area, easy operation, high absorption capacity, and photocatalytic activity. They have emerged as antimicrobial agents and growth promoters. Chahardoli et al.²⁸, showed that promoting effects of TiO₂ application on the growth of plants are concentration-dependent. Indeed, in low concentrations (50 and 100 mg L⁻¹) TiO₂ nanoparticles had a stimulating effect; while increasing the concentration to 2500 mg L⁻¹ impose an inhibitory effect on plant growth²⁴. It has been reported that TiO₂ nanoparticles increase the rate of photosynthesis and plant immunity, leading to a 30% increase in yield¹¹. In the study of Hossein et al.²⁹, foliar application of 2.5 mg L⁻¹ Ti, chlorophyll pigments (chlorophyll a, b, carotenoid), plant biomass, photochemical efficiency of photosystem II (F_v/F_m) and electron transfer rate (ETR) of soybean improve under natural light conditions. Choi et al.³⁰ reported that TiO₂ foliar application affected CO₂ absorption and chlorophyll fluorescence of cherry tomato plants. Foliar spraying of TiO₂ during cloudy days with low light intensity increased the electron transfer rate from Q_A to Q_B in the reaction center of photosystem II (ET₀/RC) and carbon dioxide (CO₂) stabilization. The use of TiO₂ nanoparticles for strawberry cultivation in the winter, which is characterized by low light intensity, increased the chlorophyll content, fruit yield, and firmness of strawberry fruit²⁰.

Controlled environment agriculture (CEA) has emerged as a new sustainable approach to increase the productivity of crops irrespective of natural environmental variations²⁷. However, increasing the efficacy of artificial light in CEA is of vital importance to make it energy-cost effective³¹. There is scarce information on the interaction of light intensity and TiO₂ nanoparticles in the CEA. Considering that TiO₂ nanoparticles have photocatalytic activity, they can be capable of increasing the efficiency of light use³². Therefore, detailed information on their practical impacts on photosynthesis and crop growth is needed.

The aim of this study was to investigate for the first time the joint inputs of cultivation light level and TiO₂ nanoparticles application on determining photosynthetic efficiency, biomass allocation and yield in controlled-environment agriculture. Radish (Raphanus raphanistrum L.) was employed as a model species, owing to the high harvest index (i.e., edible leaves and tuber), and short growth cycle³³. The obtained data serve as a conceptual basis for understanding the approaches that plants utilized under different scenarios of light energy inputs, as well as for the stimulation of photosynthetic capacity with the aim of maximizing plant growth and productivity, especially in indoor environments where light use efficiency is of obvious concern.

Results

Characterization of TiO₂

The surface morphology of TiO₂ nanoparticles was determined by scanning electron microscope (SEM) and transmission electron microscopy (TEM) (Fig. 1A–D). Briefly, TiO₂ nanoparticles were spherical or rod-shaped with -32 mV zeta potential and good dispersion. The size of most nanoparticles was in the range of 10–25 (average 18 nm) nm. Using BET method, SSA, pore volume, and average pore diameter were evaluated as 69.679 m²g⁻¹, 0.2588 cm³g⁻¹, and 14.857 nm, respectively.

Figure 1.

Representative scanning electron microscope (SEM), transmission electron microscopy (TEM) images (A–D), X-ray diffraction (XRD) pattern (E), and fourier-transform infrared spectroscopy (F) of titanium dioxide (TiO₂) nanoparticles employed in the present study.

X-ray diffraction showed all diffraction peaks of the anatase phase well (Fig. 1E). The characteristic peak of the anatase phase was 2Ɵ = 25.3³⁴. Several sharp peaks are observed in the 2Ɵ range at 20–80°. The sharp peaks in the 2Ɵ range were 25.12°, 38.25°, 48.10°, 54°, 55.1°, 63°, 69.0°, 70.50°, and 75.10°.

The FTIR spectrum of TiO₂ nanoparticles is shown in Fig. 1F. The used spectrometer has measured vibrations in the wavelength range of 350–4000 cm⁻¹. The broad peak at 3411.46 cm⁻¹ was related to the intermolecular interaction of the hydroxyl group (‒OH) attached to Ti. The 1629.55 peak corresponds to the -OH bending vibration. The broad peak at 452.225 cm⁻¹ is assigned to the bending vibration bonds (Ti–O–Ti) in the TiO₂ lattice³⁴. The observation of the mentioned peaks in the FTIR spectrum indicates the presence of Ti in the nanoparticle structure and the formation of TiO₂ nanoparticles.

In present study, TiO₂ foliar application affected chlorophyll fluorescence parameters of radish plants under different light intensities. Our results showed that foliar spraying of TiO₂ nanoparticles reduced the negative influence of low light intensity on radish. TiO₂ treatment led to an increase in EI₀/RC under low light conditions, which indicates that TiO₂ improves the rate of electron transfer flux in photosystem II reaction centers. In addition, in the reaction center of TiO₂-treated plants, the dissipated energy flux DI₀/RC decreased, indicating that TiO₂ contributed to the photosynthetic light response.

Increasing cultivation light intensity improved plant growth, and triggered biomass allocation towards the tuber in a TiO₂ concentration-dependent manner

Plants were cultivated under four light intensities (75, 150, 300 and 600 μmol m⁻² s⁻¹ PPFD), and weekly sprayed with three TiO₂ nanoparticle levels (0, 50, and 100 μmol L⁻¹).

An increase in cultivation light intensity from 75 to 150 μmol m⁻² s⁻¹ PPFD resulted in a two-fold plant leaf area (Table 1; see also Fig. 2). This increase was mostly due to larger individual leaf area, and secondarily due to an increase in leaf number. A two-step increase in cultivation light intensity from 150 (though 300) to 600 μmol m⁻² s⁻¹ PPFD led to a smaller increase in plant leaf area. This increase was exclusively related to an increase in leaf number.

Table 1.

Effect of cultivation light intensity and spray treatment (once a week, and three times in total) with titanium dioxide (TiO₂) nanoparticles on growth and morphology of radish cv.

Light intensity (µmol m−2 s−1)	TiO2 (µmol L−1)	Leaf			Petiole length (leaf −1)	Dry weight (g)			Tuber volume (mL)	SLA (cm2 g−1)	LMR (g g−1)	TMR (g g−1)
		Number (plant−1)	Area (cm2 leaf−1)	Area (cm2 plant−1)		Shoot	Tuber	Plant
75	0	4.0d	11.0d	43.9f	7.11a	0.09e	0.07f	0.15f	1.50d	520a	0.56ab	0.44de
	50	4.0d	17.9abc	71.5e	5.47bcd	0.16d	0.11f	0.27ef	1.00d	447ab	0.60a	0.40e
	100	4.0d	16.8c	67.3e	5.88bc	0.17d	0.13f	0.30ef	1.00d	403b	0.56ab	0.44de
150	0	4.7bcd	18.3abc	85.2cd	6.37ab	0.22cd	0.21ef	0.43e	2.00d	415b	0.50bc	0.50cd
	50	5.3ab	20.8abc	109.8a	4.93cde	0.42b	0.37de	0.79d	9.50c	262c	0.53ab	0.47de
	100	4.3cd	21.9ab	93.4bc	7.79a	0.25c	0.57d	0.81d	2.50d	383b	0.30d	0.70b
300	0	5.7a	16.7c	94.0bc	4.91cde	0.43b	0.56d	0.99d	2.00d	223cde	0.43c	0.57c
	50	5.0abc	21.9ab	110a	3.92ef	0.43b	0.89c	1.31c	9.50c	258cd	0.33d	0.67b
	100	5.0abc	22.6a	110a	3.77ef	0.41b	1.11b	1.52c	8.50c	271c	0.27de	0.73ab
600	0	5.7a	17.8bc	100ab	4.39def	0.55a	1.32b	1.86b	19.0b	186de	0.30d	0.70b
	50	5.3ab	19.4abc	103ab	3.29gf	0.57a	1.58a	2.14a	25.0a	182e	0.27de	0.73ab
	100	4.3cd	19.0abc	76.9de	2.17g	0.44b	1.74a	2.18a	19.0b	177e	0.20e	0.80a

Cherry belle plants. Six replicate plants were assessed per treatment. In traits, where the interaction of the two factors (light intensity, TiO₂ nanoparticle concentration) was significant, different letters indicate significant differences.

LMR leaf mass ratio, SLA specific leaf area, TMR tuber mass ratio.

Figure 2.

The growth environment (top panels), and representative images of radish cv. Cherry belle plants cultivated under different light intensities and sprayed (once a week, and three times in total) with titanium dioxide (TiO₂) nanoparticles at different levels.

At 75, 150, and 300 μmol m⁻² s⁻¹ PPFD, TiO₂ nanoparticle spray stimulated plant leaf area (Table 1; see also Fig. 2). This increase was exclusively related to an increase in individual leaf area. At 150 μmol m⁻² s⁻¹ PPFD, the low TiO₂ nanoparticle level (50 μmol L⁻¹) was optimal for plant leaf area, while at 75 and 300 μmol m⁻² s⁻¹ PPFD no significant difference was noted among the two TiO₂ nanoparticle levels. At 600 μmol m⁻² s⁻¹ PPFD, the low TiO₂ nanoparticle level (50 μmol L⁻¹) did not affect plant leaf area, while the high TiO₂ nanoparticle level (100 μmol L⁻¹) decreased it. This decrease was exclusively related to a decrease in leaf number.

Increasing cultivation light intensity resulted in shorter leaf petioles (Table 1). Within each light intensity, TiO₂ nanoparticle spray decreased leaf petiole in a concentration-dependent manner in all cases, besides one (150 μmol m⁻² s⁻¹ PPFD, 100 μmol L⁻¹).

Increasing cultivation light intensity strongly stimulated plant dry weight (Table 1; see also Fig. 2). For instance, plant dry weight was 0.15 g at 75 μmol m⁻² s⁻¹ PPFD, while it was 1.86 g at 600 μmol m⁻² s⁻¹ PPFD. This increase in plant dry weight was mediated via respective increases in both shoot and tuber dry weights.

At each cultivation light intensity, TiO₂ nanoparticle spray stimulated plant dry weight (Table 1; see also Fig. 2). No significant difference was noted between the two TiO₂ nanoparticle levels.

Increasing cultivation light intensity generally led to thinner leaves (i.e., lower SLA; Table 1). At 75 μmol m⁻² s⁻¹ PPFD, TiO₂ nanoparticle spray was also associated with thinner leaves (Table 1). At 300 μmol m⁻² s⁻¹ PPFD, the opposite trend was noted (Table 1).

Increasing cultivation light intensity generally decreased dry mass allocation to leaves, and increased dry mass allocation to the tuber (Table 1; Fig. 3). At 300 and 600 μmol m⁻² s⁻¹ PPFD, this trend was magnified by TiO₂ nanoparticle spray in a TiO₂ concentration-dependent manner (Table 1; Fig. 3).

Figure 3.

Effect of cultivation light intensity (75, 150, 300 and 600 µmol m⁻² s⁻¹) and spray treatment (once a week, and three times in total) with titanium dioxide (TiO₂) nanoparticles (0, 50 and 100 µmol L⁻¹) on biomass partitioning of radish cv. Cherry belle plants. Six replicate plants were assessed per treatment.

Increasing cultivation light intensity generally improved leaf chlorophyll and carotenoid contents in a TiO₂ concentration-dependent manner

Increasing cultivation light intensity up to 300 μmol m⁻² s⁻¹ PPFD led to an increase in leaf chlorophyll and carotenoid contents, while a further increase to 600 μmol m⁻² s⁻¹ PPFD resulted in a respective decrease (Fig. 4).

Figure 4.

Effect of cultivation light intensity (75, 150, 300 and 600 µmol m⁻² s⁻¹) and spray treatment (once a week, and three times in total) with titanium dioxide (TiO₂) nanoparticles (open, grey, and dark grey for 0, 50 and 100 µmol L⁻¹, respectively) on leaf chlorophyll, and carotenoid contents of radish cv. Cherry belle plants. Six replicate plants were assessed per treatment. Bars represent SEM. In traits, where the interaction of the two factors (light intensity, TiO₂ nanoparticle level) was significant, different letters indicate significant differences. FW, fresh weight.

TiO₂ nanoparticle spray generally stimulated leaf chlorophyll content, and counteracted the noted decrease at 600 μmol m⁻² s⁻¹ PPFD (Fig. 4A).

At 75, and 600 µmol m⁻² s⁻¹ PPFD, TiO₂ nanoparticle spray stimulated leaf carotenoid content, and counteracted the noted decrease at 600 μmol m⁻² s⁻¹ PPFD (Fig. 4B). At 150, and 300 µmol m⁻² s⁻¹ PPFD, the high TiO₂ nanoparticle level (100 μmol L⁻¹) tended to decrease leaf carotenoid content, with this effect being only significant at 300 µmol m⁻² s⁻¹ PPFD.

Increasing cultivation light intensity and applying TiO₂ nanoparticles generally induce accumulation of soluble carbohydrates in both leaves and tuber

Increasing cultivation light intensity above 150 μmol m⁻² s⁻¹ PPFD tended to increase leaf total soluble carbohydrates content, with this effect being only significant at 600 µmol m⁻² s⁻¹ PPFD (Fig. 5A). At 300, and 600 µmol m⁻² s⁻¹ PPFD, the high TiO₂ nanoparticle level (100 μmol L⁻¹) stimulated leaf total soluble carbohydrates content.

Figure 5.

Effect of cultivation light intensity (75, 150, 300 and 600 µmol m⁻² s⁻¹) and spray treatment (once a week, and three times in total) with titanium dioxide (TiO₂) nanoparticles (open, grey, and dark grey for 0, 50 and 100 µmol L⁻¹, respectively) on total carbohydrates content in leaves and tuber (A,B, respectively) of radish cv. Cherry belle plants. Six replicate plants were assessed per treatment. Bars represent SEM. In traits, where the interaction of the two factors (light intensity, TiO₂ nanoparticle level) was significant, different letters indicate significant differences. The difference in the y-axis scale among panels ought to be noted. FW, fresh weight.

Increasing cultivation light intensity enhanced tuber total soluble carbohydrates content (Fig. 5B). At 75, 150, and 300 µmol m⁻² s⁻¹ PPFD, TiO₂ nanoparticle spray generally stimulated tuber total soluble carbohydrates content.

TiO₂ nanoparticle application up-regulated photosynthetic functionality under low cultivation light intensity, while down-regulated photosynthetic functionality under high cultivation light intensity

At the lowest cultivation intensity (75 µmol m⁻² s⁻¹ PPFD), the lowest F_v/F_m was noted (Fig. 6). At the highest cultivation intensity (600 µmol m⁻² s⁻¹ PPFD), intermediate F_v/F_m values were apparent, with the highest ones being noted at 150, and 300 µmol m⁻² s⁻¹ PPFD.

Figure 6.

Representative image of the maximal quantum yield of PSII photochemistry (F_v/F_m) (equation and explanation in Table 3) of radish cv. Cherry belle plants cultivated under different light intensities (75, 150, 300 and 600 µmol m⁻² s⁻¹) and sprayed (once a week, and three times in total) with titanium dioxide (TiO₂) nanoparticles at different concentrations (0, 50 and 100 µmol L⁻¹).

At 75, and 150 µmol m⁻² s⁻¹ PPFD, TiO₂ nanoparticle spray stimulated F_v/F_m (Fig. 6). At 300 µmol m⁻² s⁻¹ PPFD, the high TiO₂ nanoparticle level (100 μmol L⁻¹) promoted F_v/F_m. At 600 µmol m⁻² s⁻¹ PPFD, TiO₂ nanoparticle spray decreased F_v/F_m.

Increasing the cultivation light intensity generally decreased the minimum fluorescence when all PSII reaction centers are open (F₀; Fig. 7A). At 75 µmol m⁻² s⁻¹ PPFD, TiO₂ nanoparticle spray decreased F₀.

Figure 7.

Effect of cultivation light intensity (75, 150, 300 and 600 µmol m⁻² s⁻¹) and spray treatment (once a week, and three times in total) with titanium dioxide (TiO₂) nanoparticles (open, grey, and dark grey for 0, 50 and 100 µmol L⁻¹, respectively) on minimum fluorescence when all PSII reaction centers are open (F₀; A), maximum fluorescence when all PSII reaction centers are closed (F_m; B), variable fluorescence of the dark-adapted sample (F_v; C) and maximal quantum yield of PSII photochemistry (F_v/F_m; D) (equations and explanations in Table 3) of radish cv. Cherry belle plants. Nine replicate plants were assessed per treatment. Bars represent SEM. In traits, where the interaction of the two factors (light intensity, TiO₂ nanoparticle level) was significant, different letters indicate significant differences.

Increasing the cultivation light intensity generally decreased the maximum fluorescence when all PSII reaction centers are closed (F_m; Fig. 7B) and the variable fluorescence of the dark-adapted sample (F_v; Fig. 7C). Within each light intensity regime, TiO₂ nanoparticle spray did not have any effect on F_m and F_v.

F_v/F_m was higher at 150 and 300 µmol m⁻² s⁻¹ PPFD, as compared to 75 µmol m⁻² s⁻¹ PPFD (Fig. 7D). TiO₂ nanoparticle spray increased F_v/F_m at 75 µmol m⁻² s⁻¹ PPFD.

Cultivation light intensity did not affect the quantum yield of electron transport (φ_E0; Fig. 8A) and the performance index in light absorption basis (PI_ABS; Fig. 8D). TiO₂ nanoparticle spray increased φ_E0 and PI_ABS at 75 µmol m⁻² s⁻¹ PPFD.

Figure 8.

Effect of cultivation light intensity (75, 150, 300 and 600 µmol m⁻² s⁻¹) and spray treatment (once a week, and three times in total) with titanium dioxide (TiO₂) nanoparticles (open, grey, and dark grey for 0, 50 and 100 µmol L⁻¹, respectively) on quantum yield of electron transport (φ_E0; A), quantum yield of energy dissipation (φ_D0; B), quantum yield for primary photochemistry (φ_PAV; C), performance index in light absorption basis (PI_ABS; D) (equations and explanations in Table 3) of radish cv. Cherry belle plants. Nine replicate plants were assessed per treatment. Bars represent SEM. In traits, where the interaction of the two factors (light intensity, TiO₂ nanoparticle level) was significant, different letters indicate significant differences.

The quantum yield of energy dissipation (φ_D0) was higher at 75 µmol m⁻² s⁻¹ PPFD as compared to 150 and 300 µmol m⁻² s⁻¹ PPFD (Fig. 8B). TiO₂ nanoparticle spray decreased φ_D0 at 75 µmol m⁻² s⁻¹ PPFD.

Increasing the cultivation intensity generally decreased the quantum yield for primary photochemistry (φ_PAV; Fig. 8C). TiO₂ nanoparticle spray decreased φ_PAV at 75 µmol m⁻² s⁻¹ PPFD.

Increasing cultivation light intensity generally decreased the specific energy fluxes per reaction center for energy absorption (ABS/RC; Fig. 9A), and the trapped energy flux per reaction center (TR₀/RC; Fig. 9B). TiO₂ nanoparticle spray decreased ABS/RC at 75 µmol m⁻² s⁻¹ PPFD (Fig. 9A).

Figure 9.

Effect of cultivation light intensity (75, 150, 300 and 600 µmol m⁻² s⁻¹) and spray treatment (once a week, and three times in total) with titanium dioxide (TiO₂) nanoparticles (open, grey, and dark grey for 0, 50 and 100 µmol L⁻¹, respectively) on specific energy fluxes per reaction center for energy absorption (ABS/RC; A), trapped energy flux per reaction center (TR₀/RC; B), electron transport flux per reaction center (ET₀/RC; C), dissipated energy flux (DI₀/RC; D) (equations and explanations in Table 3) of radish cv. Cherry belle plants. Nine replicate plants were assessed per treatment. Bars represent SEM. In traits, where the interaction of the two factors (light intensity, TiO₂ nanoparticle level) was significant, different letters indicate significant differences.

The electron transport flux per reaction center (ET₀/RC) was lower at 600 µmol m⁻² s⁻¹ PPFD as compared to lower intensities (Fig. 9C). TiO₂ nanoparticle spray did not affect ET₀/RC.

The dissipated energy flux (DI₀/RC) was higher at 75 µmol m⁻² s⁻¹ PPFD as compared to higher light intensities (Fig. 9D). TiO₂ nanoparticle spray decreased DI₀/RC at 75 µmol m⁻² s⁻¹ PPFD (Fig. 9D). At 0 µmol m⁻² s⁻¹ PPDF measuring light intensity, ΦPSII was not affected by cultivation light intensity (Fig. 10A). As measuring light intensity increased, ΦPSII decreased. Leaves cultivated under higher light intensities generally sustained higher ΦPSII values across measurement light levels (100−1000 μmol m⁻² s⁻¹ PPFD). At 75 µmol m⁻² s⁻¹ PPFD during cultivation, the high TiO2 nanoparticle level (100 μmol L−1) tended to sustain higher ΦPSII values. As measurement light intensity increased, rETR also increased (Fig. 10B). Differences among cultivation light intensities were magnified, as measurement light intensity increased. The lower rETR was noted in leaves cultivated under 75 µmol m−2 s−1 PPFD.

Figure 10.

Rapid light curve of effective quantum yield of PSII (Φ_PSII, A) and electron transport rate (rETR, B) (equations and explanations in Table 3) as a function of measurement light intensity in radish cv. Cherry belle plants cultivated under different light intensities (75, 150, 300 and 600 µmol m⁻² s⁻¹) and sprayed (once a week, and three times in total) with titanium dioxide (TiO₂) nanoparticles at different concentrations (0, 50 and 100 µmol L⁻¹). Nine replicate plants were assessed per treatment. Bars represent SEM.

Increasing cultivation light intensity and applying TiO₂ nanoparticles generally stimulate non-photochemical quenching

The non-photochemical quenching (NPQ) was higher at 600 µmol m⁻² s⁻¹ PPFD as compared to lower light intensities (Fig. 11). TiO₂ nanoparticle spray generally increased NPQ across cultivation light intensities. This effect was comparable at different cultivation light intensities.

Figure 11.

Effect of cultivation light intensity (75, 150, 300 and 600 µmol m⁻² s⁻¹) and spray treatment (once a week, and three times in total) with titanium dioxide (TiO₂) nanoparticles (open, grey, and dark grey for 0, 50 and 100 µmol L⁻¹, respectively) on non-photochemical quenching (equation and explanation in Table 3) of radish cv. Cherry belle plants. Nine replicate plants were assessed per treatment. Bars represent SEM. The interaction of the two factors (light intensity, TiO₂ nanoparticle level) was significant, and different letters indicate significant differences.

Discussion

In this study, the optimal concentration (50, and 100 μmol L⁻¹) of TiO₂ nanoparticles foliar application for stimulating photosynthetic efficiency, and eventually yield was evaluated in radish. There are some previous indications highlighting more effectiveness of the foliar application of TiO₂ nanoparticles on crop growth, photosynthesis, and yield. For instance, the application of TiO₂ nanoparticles (average diameter 25 nm, in the form of anatase, zeta potentials ˗22.5 mV) in aerosol format was more effective compared to TiO₂ nanoparticles as a soil amendment for increasing photosynthesis and lycopene content³⁵. The dependence of the respective promotive effect on cultivation light intensity (75, 150, 300 and 600 μmol m⁻² s⁻¹ PPFD) was also investigated in the present study.

Increasing cultivation light intensity resulted in a larger plant leaf area (Table 1; see also Fig. 2), and in this way improved light capture³⁶. The plant leaf area increase was most prominent when light intensity increased from 75 to 150 μmol m⁻² s⁻¹ PPFD, while a further increase in light intensity led to a more moderate (plant leaf area) increase. In the former case (from 75 to 150 μmol m⁻² s⁻¹ PPFD), the increase in plant leaf area was mostly due to larger individual leaf area, whereas in the latter case (≥ 300 μmol m⁻² s⁻¹ PPFD), it was exclusively due to the larger number of leaves (thus enhanced leaf initiation). Therefore, the initial light level (rather than the percentage of increase) strongly determines both the promoting effect of increasing light intensity on plant leaf area, and the underlying process by which this increase is realized (i.e., individual leaf area and/ or the number of leaves).

When cultivation light intensity did not exceed 300 μmol m⁻² s⁻¹ PPFD, TiO₂ nanoparticle spray also stimulated plant leaf area (Table 1; see also Fig. 2). The TiO₂ nanoparticle spray-induced increase in plant leaf area was due to increased individual leaf area. The TiO₂ nanoparticle spray-induced increase in plant leaf area was more prominent as cultivation light intensity was lower. Therefore, applying TiO₂ nanoparticles provides an additional advantage in plants cultivated at low light levels.

Increasing cultivation light intensity resulted in larger plant biomass (Table 1; see also Fig. 2). The applied light levels (75–600 m⁻² s⁻¹ PPFD) were thus below the light saturation point of the crop¹². The light intensity-induced increase in plant biomass was more prominent as the initial light intensity was lower. Across cultivation light intensities, TiO₂ nanoparticle spray stimulated plant biomass (Table 1; see also Fig. 2). The TiO₂ nanoparticle application-induced increase in plant biomass was also more prominent as the cultivation light intensity was lower. Both TiO₂ nanoparticle levels (50, and 100 μmol L⁻¹) were equally effective in stimulating plant biomass.

More plant mass was allocated to the leaves (i.e., higher LMR), as cultivation light intensity was lower (Table 1; Fig. 3). Thus, plants favored biomass partitioning to the light-intercepting organ (leaves) to amplify carbon gain under low light level conditions, whereas this partitioning gradually shifted to the tuber, which is evidently affiliated with the supply of other resources (i.e., water and nutrients), under higher light level circumstances¹². Fine-tuning of leaf thickness (SLA) by regulating the light capture surface was another important aspect of adaptation to the light level in radish. Leaves were consistently thinner (i.e., higher SLA) as the light level decreased (Table 1). Increasing SLA stimulates the amount of light which is intercepted, and in this way maximizes carbon assimilation. This adaptation in leaf morphology is common in low-light situations, and has been reported in several taxa¹².

Throughout the distribution chain of edible greens, the intensity of leaf greenness is an index of quality⁵. At inadequate leaf chlorophyll level, photosynthetic efficiency is also impeded²³. Carotenoids are the main antioxidant metabolites, contributing to plant defense, and exhibiting health-promoting properties when consumed⁵. Increasing cultivation light intensity up to 300 μmol m⁻² s⁻¹ PPFD improved both leaf chlorophyll and carotenoid contents, while a further increase (600 μmol m⁻² s⁻¹ PPFD) exerted an adverse effect (Fig. 4). At 75, 150, and 600 μmol m⁻² s⁻¹ PPFD, TiO₂ nanoparticle spray promoted leaf chlorophyll content (Fig. 4A). At 75, and 600 μmol m⁻² s⁻¹ PPFD, TiO₂ nanoparticle spray also enhanced leaf carotenoid content (Fig. 4B). In either case, the optimum TiO₂ nanoparticle concentration depended on light level. Therefore, applying TiO₂ nanoparticles improves both leaf chlorophyll and carotenoid contents, especially under extreme light environments (i.e., low or very high).

Increasing cultivation light intensity generally stimulated the content of total soluble carbohydrates (primary energy reserves) in both leaves and tuber, with this effect being more prominent in the tuber (Fig. 5). TiO₂ nanoparticle spray further improved both leaf and tuber total soluble carbohydrates contents, with this effect being more pronounced in the leaf (Fig. 5). The level of primary energy reserves has been earlier associated with prolonged postharvest longevity^22,37. For instance, an increased content of soluble carbohydrates may support maintenance needs (by sustaining ATP synthesis), and protect membrane integrity (by scavenging reactive oxygen species)^22,37.

Growth environment determines the light energy absorbance by the PSII apparatus, and this is readily reflected by chlorophyll fluorescence analysis³⁸. As growth PPFD increased, a decreasing trend was noted in the F₀, F_m, and F_v (Fig. 7A–C). These parameters (F₀, F_m, and F_v) were generally not affected by TiO₂ nanoparticle spray, except for a decrease in F₀ at 75 µmol m⁻² s⁻¹ PPFD. The decrease in F₀ may be associated with a reduction in plastoquinone electron receptors and incomplete oxidation due to retardation, which results in electron transfer chain postponement in PSII. Alternatively, F₀ may be decreased by the separation of light-harvesting Chl a/b protein complexes in PSII³⁹. The reduction in F_m is probably related to the deactivation of proteins in chlorophyll structure.

Higher values of F_v/F_m [(F_m – F₀)/F_m; Table 3] reflect a better light use efficiency³⁹. Under stress, both the rising trend of F₀ and the declining trend of F_m typically underlie the decrease in F_v/F_m^15,25,40. This decline in F_v/F_m denotes a stress-induced damage in the structure or function of PSII. As growth PPFD increased up to 300 µmol m⁻² s⁻¹ PPFD, F_v/F_m also increased (Fig. 7D; see also Fig. 6). A further increase in growth PPFD to 600 µmol m⁻² s⁻¹ led to a decrease in F_v/F_m. Therefore, the optimum growth light intensity for light use efficiency was clearly the 300 µmol m⁻² s⁻¹ PPFD. In lettuce, the optimum growth light intensity was 600 µmol m⁻² s⁻¹ PPFD^{16,17,41–44}, while for Aloe vera L. it was 50% of sunlight as compared to 75 or 100%⁴⁴. F_v/F_m was generally not affected by TiO₂ nanoparticle spray, except for an increase noted at 75 µmol m⁻² s⁻¹ PPFD. Therefore, TiO₂ nanoparticle spray improved light use efficiency at the lowest growth light intensity.

Table 3.

Abbreviations, definitions and formulas of the chlorophyll fluorescence parameters assessed in the current study.

Category	Abbreviation	Definition	Equation
Basic parameters	F0	Minimum fluorescence, when all PSII reaction centers are open (O-step of OJIP transient)	F50µs
	FJ	Fluorescence intensity at the J-step (2 ms) of OJIP	F2ms
	FI	Fluorescence intensity at the I-step (30 ms) of OJIP	F30ms
Fluorescence parameters	Fm	Maximum fluorescence, when all PSII reaction centers are closed (P-step of OJIP transient)	F1s = Fp
	Fv	Variable fluorescence of the dark-adapted sample	Fm – F0
	VJ	Relative variable fluorescence at time 2 ms (J-step) after start of actinic light pulse	(FJ – F0)/(Fm – F0)
	VI	Relative variable fluorescence at time 30 ms (I-step) after start of actinic light pulse	(F30ms – F0)/(Fm – F0)
	Fv/Fm	Maximal quantum yield of PSII photochemistry	1 – (F0/Fm) = (Fm – F0)/Fm = φP0 = TR0/ABS
Quantum yields and efficiencies/probabilities	φE0	The quantum yield of electron transport	[1– ( F0/ Fm)](1 – VJ) = ET0/ABS
	φD0	Quantum yield of energy dissipation	F0/Fm
	φPAV	Average (from time 0 to tFM) quantum yield for primary photochemistry	φP0 (1 − VJ) = φP0 (SM/tFM)
Specific energy fluxes (per QA reducing PSII reaction center)	ABS/RC	The specific energy fluxes per reaction center for energy absorption	M0 (1/VJ)(1/φP0)
	TR0/RC	Trapped energy flux (leading to QA reduction) per reaction center	M0 (1/VJ)
	ET0/RC	Electron transport flux (further than QA−) per reaction center	M0 (1/VJ)(1 − VJ)
	DI0/RC	Dissipated energy flux	(ABS/RC) − (TR0/RC)
Performance indices (products of terms expressing partial potentials at steps of energy bifurcations)	PIABS	Performance index for the photochemical activity	[(γRC/1 − γRC)( φP0 /1 − φP0)(ψE0 /1 − ψE0)]
	NPQ	Non-photochemical quenching	(Fm/Fm´) – 1
Rapid light curve as a function of PPFD	ΦPSII	Effective quantum yield of PSII	∆F/Fm´ = (Fm´ – F)/ Fm´
	rETR	Relative electron transport rate	ΦPSII × PPFD × 0.5 × 0.84

PI_ABS is an index of the quantum yield of absorbed photons, and is highly sensitive to changes in the concentration of reaction centers, initial photochemistry, and electron transport^14,45. Exposure to non-optimal PPFD leads to alterations in absorption and energy trapping, thereby adversely influencing PI_ABS¹⁷. Similarly to F_v/F_m, PI_ABS increased as growth PPFD increased up to 300 µmol m⁻² s⁻¹ PPFD, and then (600 µmol m⁻² s⁻¹ PPFD) slightly decreased (Fig. 8D). Likewise to F_v/F_m, TiO₂ nanoparticle spray increased PI_ABS at 75 µmol m⁻² s⁻¹ PPFD. A decrease in PI_ABS may be due to the higher light energy absorption, dissipated energy flux, and lower electron transport per reaction center (expressed by ABS/RC, DI₀/RC, and ET₀/RC, respectively)⁴⁶.

ABS/RC flux is divided into TR₀/RC, ET₀/RC, and DI₀/RC, while the share of ABS is converted to DI and TR. As growth PPFD increased, ABS/RC tended to decrease (Fig. 9A). ABS/RC was generally not affected by TiO₂ nanoparticle spray, except for a decrease apparent at 75 µmol m⁻² s⁻¹ PPFD. The decreasing trend in ABS/RC indicates the more efficient function of the electron transport system, which decreases the excitation force on each reaction center and directs that energy mostly toward photosystem I.

At 600 µmol m⁻² s⁻¹ PPFD, ET₀/RC was lower (Fig. 9C), indicating an adverse effect in the rate of electron transport flux on PSII reaction centers. Increasing cultivation light intensity generally decreased TR₀/RC (Fig. 9B). At 75 µmol m⁻² s⁻¹ PPFD, DI₀/RC was higher (Fig. 9D). These parameters (ET₀/RC, TR₀/RC, and DI₀/RC) were generally not affected by TiO₂ nanoparticle spray, except a decrease in DI₀/RC at 75 µmol m⁻² s⁻¹ PPFD. A high DI₀/RC is generally associated with decreased F_v/F_m and an increased incidence of photoinhibition^17,47. Since DI₀/RC is related to the partial deactivation of PSII reaction centers, it usually increases when PSII reaction centers cannot transfer energy upstream of PSII as a consequence of damage to the thylakoid membrane^14,48. Shabbir et al.⁴⁹ reported that the photochemical efficiency of PSII increased by applying 90 mg L⁻¹ TiO₂ to vetiver plants. In the study by Gao et al.⁵⁰ treatment of spinach with TiO₂ resulted in the induction of a complex of Rubisco and Rubisco activase to accelerate the carboxylation of Rubisco and ultimately improve photosynthetic efficiency. In the study of Azmat et al.⁵¹, the treatment of spinach with TiO₂ nanoparticles increased the starch content due to its photocatalytic properties.

The absorbed light energy is quenched via photochemistry, fluorescence, or heat dissipation. In a healthy photosystem, a large portion is used to perform photosynthesis (the so-called photochemical quenching), and a trivial one is released via fluorescence. The third fate is NPQ, in which the excess excitation energy of the chlorophyll in the PSII complex is harmlessly dissipated into thermal energy⁵². Therefore, NPQ signifies an effective way for dissipating excessive irradiation into heat by the photosynthetic apparatus⁵³. Studying the behavior of NPQ provides information regarding the xanthophyll cycle activity and other energy-quenching pathways which are induced by the proton concentration inside thylakoids^54–56. NPQ was higher at 600 µmol m⁻² s⁻¹ PPFD (Fig. 11). Across cultivation light intensities, TiO₂ nanoparticle spray generally increased NPQ. Thus, it can be concluded that plants grown under the highest PPFD and/ or TiO₂ nanoparticle spray directed more efficiently the excess excitation energy through non-radiative dissipative processes. This high energy input can be due to feedback inhibition of carbohydrate accumulation on the electron transport chain. An increase in the level of carotenoids as a result of exposure to higher PPFDs or TiO₂ concentrations is a helpful strategy to direct the energy input toward the NPQ. In this way, the photosynthetic apparatus remains adequately protected under conditions of excessively high light energy input.

The rapid light curves illustrate the short-term photosynthetic response to rising light levels. Studying them is a proper way to gain knowledge on overall photosynthetic performance as well as the saturation characteristics of electron transport⁴⁹. In the present study, rapid light curves of both Φ_PSII and rETR were assessed. As measuring light intensity increased, Φ_PSII decreased whereas rETR increased (Fig. 10). The decline in Φ_PSII denotes a limited capacity for photochemical energy usage. In rETR, both the lower increase and the development of a plateau depict the light intensity, where the photosynthetic pathway became limited. Across measurement light levels (100 − 1000 μmol m⁻² s⁻¹ PPFD), leaves cultivated under higher light intensities generally sustained higher Φ_PSII and rETR values (Fig. 10). At 75 µmol m⁻² s⁻¹ PPFD during cultivation, TiO₂ nanoparticle spray (100 μmol L⁻¹) tended to sustain higher Φ_PSII and rETR values. In accordance, it has been shown that foliar spraying of 100 mg L⁻¹ of TiO₂ nanoparticles under low light intensity increased the rate of photosynthesis and yield in cherry tomato plants³⁰.

In conclusion, radish plants employed two mechanisms under contrasting lighting conditions. In the first mechanism (morphological adaptation), under low light, plants partitioned their biomass toward upper-ground parts which resulted in smaller tubers. Furthermore, plants allocated their biomass toward leaf expansion rather than leaf thickness which was indicated by the high SLA of plants under low light intensity (shade avoidance response). Conversely, plants under high light partitioned most of biomass toward underground parts which resulted in production of bigger tubers. They also produced thicker leaves with limited area to avoid capturing excessive light photons. In the second mechanism (photosynthetic acclimation), plants decreased NPQ and accumulated less carbohydrates under low light condition, while increased NPQ and produced more carbohydrates under high light condition. TiO₂ nanoparticle application mitigate the shade avoidance response under low light intensities, while increased NPQ and reduced electron transport under high light due to higher energy perception (Fig. 12).

Figure 12.

A schematic illustration on the effect of low and high cultivation light intensity and titanium dioxide (TiO₂) nanoparticles application on radish plants. Under low light, plants partitioned less biomass towards the tubers and allocated their biomass toward leaf expansion rather than leaf thickness. Low light-exposed plants decreased NPQ and accumulated less carbohydrates. Conversely, under high light, plants partitioned most of their biomass into the tubers and produced thick leaves with limited area. They increased NPQ and produced more carbohydrates under high light condition. TiO₂ nanoparticle application directed more biomass into the tubers under low light intensities, while increased NPQ under high light condition.

Materials and methods

Plant material and growth conditions

Seeds of radish (R. raphanistrum) cv. Cherry Belle were sterilized (3 min) in sodium hypochlorite solution (5%; v/v), and then rinsed (3 min) with distilled water. Seeds were then manually planted in a seedling tray (30 × 18 × 5 cm) filled with a mixture of peat and perlite (1:1, v/v). The sowing depth was kept constant, and seeds were covered with substrate immediately after sowing. At the two-leaf stage, plants were transferred to 2 L pots [diameter (top) × diameter (bottom) × height = 15 × 13 × 15 cm] containing the same mixture. To facilitate drainage, 5 ± 1 g of pebbles (particle size of 16–30 mm) had been earlier placed at the bottom of each pot. Next, 120 pots were equally distributed to four environmentally-controlled growth chambers (l × w × h = 1.5 × 1.0 × 1.3 m) for realizing treatments. A density of 20 plants per m² was employed.

Twelve factors (4 light intensities × 3 TiO₂ nanoparticle spray treatments) were applied as a factorial experiment based on a completely randomized design. Each growth chamber included a single light intensity level, which was set at 75, 150, 300 and 600 μmol m⁻² s⁻¹ PPFD for 16 h d⁻¹ (06:00–22:00), respectively. In all chambers, light was provided by red (660 nm peak wavelength) and blue (450 nm peak wavelength) LED modules (Parcham Co, Tehran, Iran) with a (red to blue) ratio of 3 to 1. Light intensity was determined by a handy fluorometer (PAR-FluorPen FP 100-MAX, Photon Systems Instruments, Drásov, Czech Republic), while the spectrum was defined by a handy spectrometer (Seconic C7000, Japan).

The remaining environmental variables were identical among the four growth chambers. Day/ night air temperature was set to 26/ 16 °C, and relative air humidity was adjusted to 65–70%. Two ventilation fans (12 V, 0.90A) were installed in each growth chamber to ensure uniform air circulation. Plants were cultivated in a customized liquid culture hydroponic system, by using a modified Hoagland nutrient solution (pH ≈ 5.8; EC ≈ 1.8 dS m⁻¹; detailed composition in Table 2). Growth media moisture was maintained at or near maximum water-holding capacity. To prevent salt accumulation, pots were also irrigated with distilled water twice a week (> 20% drainage).

Table 2.

Stock solutions' ingredients employed in the present study. From each stock solution, 10 mL was added to prepare 1 L of nutrient solution.

Ingredient	Quantity for 1 L of stock solution (g)
Calcium nitrate	97
Potassium nitrate	61
Magnesium sulfate (16% MgO)	25
Mono-potassium phosphate	19
Potassium sulfate	17
Iron EDDHA (6%)	3.723
Mono-ammonium phosphate	1
Borax (11.3% B)	0.383
Zinc sulfate	0.35
Manganese sulfate	0.3
Copper sulfate	0.04
Sodium molybdate (39.6%)	0.024

Plant- and leaf-level measurements were conducted. For leaf-level measurements, sampled leaves had grown under direct light, and were fully-expanded. Replicate leaves were selected from separate plants. To minimize border effects, experimental plants were surrounded by border plants (adjacent to chamber walls) that were not sampled. Samples were collected at the onset of the light period (06:00–07:00 h). Different treatments were always assessed simultaneously. In growth and biomass allocation measurements, the time between sampling and the start of the evaluation did not exceed 15 min. In the remaining evaluations, samples were placed in vials, flash frozen in liquid nitrogen, and transferred to a freezer (− 80 °C) for storage. In all cases, sampling was conducted at the end of the cultivation period, besides the chlorophyll fluorescence evaluation, which was performed 1 d earlier. In all determinations, six replicates were assessed per treatment, besides chlorophyll fluorescence assessments, where nine replicates were evaluated.

Preparation of TiO₂ suspension

The methods used for the synthesis of TiO₂ nanoparticles are Sol–gel route, fame hydrolysis, co-precipitation, impregnation and chemical vapor deposition. Three crystalline phases of titanium dioxide, are anatase (tetragonal), rutile (tetragonal), and brookite (orthorhombic), that brookite has no commercial value¹¹. TiO₂ nanoparticles in the anatase form (˃ 99% purity, APS: 10–25 nm, Color: white, Bulk density: 0.24 g/cm³, True density: 3.9 g/cm³, PH: 6–6.5) were obtained from a commercial supplier (Iranian Nanomaterial Pioneers Company, Mashhad, Iran). The shape, size and crystal structure of TiO2 nanoparticles were evaluated using SEM, TEM, XRD and FTIR. The Specific surface area (SSA) and pores were analyzed by BET (Brunauer–Emmett–Teller) instrument.

In each light intensity regime, following adaptation (3 d), TiO₂ nanoparticles at different concentrations (0, 50, and 100 μmol L⁻¹) were weekly applied (three times in total) via foliar spray. The suitable concentration range was selected based on both a comprehensive literature survey^20,28,57, and a pre-experiment. Each time before application, the TiO₂ nanoparticles solution was ultrasonically homogenized using a sonicator (UP100H, Hielscher Ultrasonics, Germany)^58–60. At the time of the first application, plants were still at the two-leaf stage.

Plant growth, morphology, and biomass allocation

The petiole (stalk) length (from the base to the leaf joint), the number of leaves, individual leaf area (one-sided surface area), and plant leaf area were first determined. For leaf area assessment, leaves were scanned (HP Scanjet G4010, Irvine, CA, USA) and then evaluated by using the Digimizer software (version 5.3.5, MedCalc Software, Ostend, Belgium)^21,61.

Following removal of the substrate from the tuber via gentle washing, tuber volume was measured by employing a volume-displacement technique^62,63. Tubers were suspended in a cylinder filled with water, and tuber volume was then determined by measuring the volume of displaced water.

Leaf and tuber (fresh and dry) masses were also recorded (± 0.01 g; MXX-412; Denver Instruments, Bohemia, NY, USA). For measuring dry weight, samples were placed in a forced-air drying oven for 72 h at 80 °C. By using dry mass, specific leaf area (SLA; leaf area/leaf mass), leaf mass ratio (LMR; leaf mass/plant mass), and tuber mass ratio (TMR; tuber mass/plant mass) were calculated.

Prior to the destructive measurements, plant images were obtained by using a digital camera (Canon EOS M2; Canon Inc., Tokyo, Japan). The plants were manually moved to the image capture station. The imaging station included top and side lighting units (fluorescent tubes; Pars Shahab Lamp Co., Tehran, Iran). The camera-to-plant distance was maintained constant. One image (RGB) was obtained per plant.

Leaf chlorophyll and carotenoid content

Samples (0.1 g) were homogenized with the addition of 10 mL of 100% acetone. The extract was then centrifuged (14,000 g for 15 min), and the supernatant was collected. Since chlorophyll is light sensitive, the extraction took place in a dark room^58,59. The obtained extract was subjected to reading on a spectrophotometer (Optizen pop, Mecasys Co. Ltd. Daejeon, Korea). Total chlorophyll and carotenoid contents were calculated according to Lichtenthaler and Wellburn⁶⁴.

Leaf total soluble carbohydrates content

Colorimetric quantification of leaf total soluble carbohydrates (i.e., reducing and non-reducing sugars) content was performed⁶⁵. Shortly before analysis (< 30 min), the anthrone reagent was prepared under darkness by dissolving 0.1 g of anthrone (0.2%) in 100 mL of concentrated sulfuric acid (98%). Sample (0.1 g) and anthrone reagent (1 mL) were loaded in tubes, which were placed in a water bath (90 °C for 15 min), cooled (0 °C for 5 min), and vortexed (1 min). Before reading, a heating step (20 min) to room temperature (25 °C) was performed. The absorbance was measured at 620 nm with a spectrophotometer (Optizen pop, Mecasys Co. Ltd. Daejeon, Korea). A standard curve based on a series of known glucose concentrations was prepared.

Maximum quantum yield of photosystem II (PSII)

As a sensitive indicator of photosynthetic performance, dark-adapted values of the maximum quantum yield of PSII (F_v/F_m; equation and explanation in Table 3) were non-invasively recorded in leaves of each treatment^22,37,63. Measurements were performed by using a portable imaging fluorometer (Handy FluorCam FC 1000-H, Photon Systems Instruments, Drásov, Czech Republic). Before taking measurements, samples were dark-adapted (≥ 20 min). Then, F_v/F_m was evaluated by a custom-made protocol^21,23, where leaves were exposed to short flashes followed by long saturated light pulses (3900 µmol m⁻² s⁻¹ PPFD) to cause a reduction in the primary quinone acceptor of PSII.

Polyphasic chlorophyll fluorescence transient (O–J–I–P) evaluation

A polyphasic chlorophyll fluorescence induction curve (O–J–I–P-transient) was obtained in the attached leaves of each treatment. By employing this test, the shape changes of the O–J–I–P transient are quantitatively translated to a set of parameters (see Table 3), which relate to the in vivo adaptive behavior of the photosynthetic apparatus (especially PSII) to the growth environment^25,26. Measurements were conducted by using a PAR- FluorPen FP 100-MAX (Photon Systems Instruments, Drásov, Czech Republic) following dark adaptation (≥ 20 min). The employed light intensity (3900 μmol m⁻² s⁻¹ PPFD) was sufficient to generate maximal fluorescence in all treatments.

Following dark adaptation, leaves exhibit a polyphasic chlorophyll fluorescence rise during the first second of illumination. The fluorescence transient, plotted on a logarithmic time scale, typically includes the following phases: O to J, J to I, and I to P. F₀ was measured at 50 μs, fluorescence intensity at J-step was assessed at 2 ms, and fluorescence intensity at I-step was evaluated at 30 ms^39,66.

Non-photochemical quenching

Another set of dark-adapted attached leaves were used to determine non-photochemical quenching (NPQ; equation and description in Table 3) by using a PAR-FluorPen FP 100-MAX (Photon Systems Instruments, Drásov, Czech Republic). Calculations were performed by using the FluorPen software (v. 7; Photon Systems Instruments, Drásov, Czech Republic).

Rapid light curve of effective quantum yield of PSII and electron transport rate

The rapid light curve of effective quantum yield of PSII (Φ_PSII; equation and description in Table 3) was determined with a handy fluorometer (PAR-FluorPen FP 100-MAX, Photon Systems Instruments, Drásov, Czech Republic). The dark-adapted leaves were exposed to a gradual increase of actinic blue light illumination in six steps (0, 100, 200, 300, 500 and 1000 μmol m⁻² s⁻¹ PPFD). Each illumination step was separated by 10 s intervals. Illuminating the saturating flash required 0.8 s. Data were analyzed by using the FluorPen software (v. 7; Photon Systems Instruments, Drásov, Czech Republic). Relative electron transport rate (rETR; equation and description in Table 3) was also calculated⁵⁴.

Statistical analysis

Data were subjected to analysis of variance using SAS software (v. 9.4, SAS Institute Inc., Cary, NC, USA). Data were firstly tested for normality (Shapiro–Wilk test) and homogeneity of variances (Levene’s test). A split-plot design was employed, with cultivation light intensity (75, 150, 300 and 600 μmol m⁻² s⁻¹ PPFD) as the main factor, and TiO₂ nanoparticles concentration (0, 50, and 100 μmol L⁻¹) as the split factor. Treatment effects were tested at a 5% probability level and mean separation was carried out using the least significant differences based on Duncan's multiple range test (P ≤ 0.05).

Abbreviations

ABS/RC

The specific energy fluxes per reaction center for energy absorption

DI₀/RC

The dissipated energy flux

ET₀/RC

The electron transport flux per reaction center

F_m

The maximum fluorescence when all PSII reaction centers are closed

F_v

The variable fluorescence of the dark-adapted sample

F_v/F_m

Ratio of variable to maximum fluorescence

FW

Fresh weight

F₀

The minimum fluorescence when all PSII reaction centers are open

LMR

Leaf mass ratio

NPQ

Non-photochemical quenching

PI_ABS

The performance index in light absorption basis

PPFD

Photosynthetic photon flux density

PSII

Photosystem II

rETR

Relative electron transport rate

SLA

Specific leaf area

TEM

Transmission electron microscopy

TiO₂

Titanium dioxide

TMR

Tuber mass ratio

TR₀/RC

The trapped energy flux per reaction center

XRD

X-ray diffraction

∆F/F_m′

Effective quantum yield of PSII

φ_D0

The quantum yield of energy dissipation

Φ_PSII

Effective quantum yield of PSII

φ_E0

The quantum yield of electron transport

φ_PAV

The quantum yield for primary photochemistry

Author contributions

Conceptualization, A.V., M.M-N, S.Al., and D.F.; data curation, A.V., S.Ab., M.M-N., and S.R.; formal analysis, A. V., M.M-N.; investigation, A. V., S.Ab., Z.M., M.M-N.; project administration, S.Al.; supervision, S.Al., S.R. and D.F.; funding, A. V, S. Al., and S.R.; visualization, M.M-N; writing—original draft, D.F.; writing—review and editing, S.Al., M.M-N., G.T., and D.F. All authors have read and agreed to the published version of the manuscript.

Funding

This research was financed by a research grant from the Ministry of Science, Research and Technology of Iran as a post-doctoral project to Akram Vatankhah (No: 5547981).

Data availability

The main data are contained within the article. Data are available upon request from the corresponding author.

Competing interests

The authors declare no competing interests.