*Article* **Influence of Light of Different Spectral Compositions on the Growth, Photosynthesis, and Expression of Light-Dependent Genes of Scots Pine Seedlings**

**Pavel Pashkovskiy 1,\*, Vladimir D. Kreslavski 2, Yury Ivanov 1, Alexandra Ivanova 1, Alexander Kartashov <sup>1</sup> , Alexander Shmarev <sup>2</sup> , Valeriya Strokina 2, Vladimir V. Kuznetsov <sup>1</sup> and Suleyman I. Allakhverdiev 1,\***


**Abstract:** Varying the spectral composition of light is one of the ways to accelerate the growth of conifers under artificial conditions for the development of technologies and to obtain sustainable seedlings required to preserve the existing areas of forests. We studied the influence of light of different quality on the growth, gas exchange, fluorescence indices of Chl *a*, and expression of key light-dependent genes of *Pinus sylvestris* L. seedlings. It was shown that in plants growing under red light (RL), the biomass of needles and root system increased by more than two and three times, respectively, compared with those of the white fluorescent light (WFL) control. At the same time, the rates of photosynthesis and respiration in RL and blue light (BL) plants were lower than those of blue red light (BRL) plants, and the difference between the rates of photosynthesis and respiration, which characterizes the carbon balance, was maximum under RL. RL influenced the number of xylem cells, activated the expression of genes involved in the transduction of cytokinin (Histidinecontaining phosphotransfer 1, *HPT1*, Type-A Response Regulators, *RR-A*) and auxin (Auxin-induced protein 1, *Aux/IAA*) signals, and reduced the expression of the gene encoding the transcription factor phytochrome-interacting factor 3 *(PIF3*). It was suggested that RL-induced activation of key genes of cytokinin and auxin signaling might indicate a phytochrome-dependent change in cytokinins and auxins activity.

**Keywords:** photomorphogenesis; *Pinus sylvestris*; light of various spectral composition; photosynthesis; chlorophyll fluorescence; gene expression; pigment content

#### **1. Introduction**

The quality of light is an important factor in regulating plant growth and development during ontogenesis, including germination, photomorphogenesis, flowering induction, etc. At the beginning of ontogenesis, most plants are forced to vegetate under shading conditions while growing underneath taller plants, which also leads to a decrease in the quality of light. Green and far-red light (FRL) predominate under the forest canopy because light in the red and blue ranges of the spectrum is effectively absorbed by the chlorophyll of taller plants. This forces the seedlings of most woody plants to adapt to indifferent light qualities [1]. For example, plants growing under a forest canopy acclimatize to a low red:far red (R:FR) ratio, which causes shoot elongation [1]. It was previously shown that blue light (BL), on the contrary, inhibits shoot growth [2,3]. This leads to plants with a high BL level and a high R:FR ratio growing low but with an increased leaf surface, which in turn affects the intensity of photosynthesis [4].

The observed changes in the global climate under all forecast scenarios suggest a decline in coniferous species in the temperate zone of Europe [5]. In this regard, the

**Citation:** Pashkovskiy, P.; Kreslavski, V.D.; Ivanov, Y.; Ivanova, A.; Kartashov, A.; Shmarev, A.; Strokina, V.; Kuznetsov, V.V.; Allakhverdiev, S.I. Influence of Light of Different Spectral Compositions on the Growth, Photosynthesis, and Expression of Light-Dependent Genes of Scots Pine Seedlings. *Cells* **2021**, *10*, 3284. https://doi.org/10.3390/cells10123284

Academic Editor: Seiji Akimoto

Received: 22 October 2021 Accepted: 22 November 2021 Published: 24 November 2021

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**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

development of technologies for obtaining sustainable seedlings is required to preserve the existing areas of coniferous forests. Varying the spectral composition of light can be one of the simplest ways to accelerate the growth of seedlings under artificial conditions [6]. However, to select the optimal spectral composition of light under artificial light conditions, it is necessary to know how the light of different spectral ranges affects the growth and photosynthetic parameters of seedlings of coniferous plants.

Plants have several types of photoreceptors that respond to changes in environmental light conditions. Under natural light, plants are simultaneously exposed to light of different wavelengths, resulting in cross-signaling of light between multiple photoreceptors. Phytochromes are among the most characterized photoreceptors. They are RL and FRL sensors and they regulate many developmental processes, including seed germination and hypocotyl growth [1]. Other well-known receptors, the cryptochromes, perceive light in the blue and UV ranges of the spectrum. They are involved in the growth processes and de-etiolation of seedlings and are also involved in circadian rhythms [7]. Photoreceptors have been studied in detail in *Arabidopsis thaliana*. They include five phytochromes (PHYA to PHYE) and two major cryptochromes (CRY1 and CRY2). In gymnosperms, PHYN is orthologous to PHYA of angiosperms, while PHYO is orthologous to PHYC [8]. In addition, gymnosperms have PHYP, which genetically occupies an intermediate position between the PHYB of *Oryza sativa* and the PHYE of *Arabidopsis thaliana* [9]. From the experiments carried out by Clapham et al., 2002, it was found that pine and spruce react to the RL/FRL ratio differently than angiosperms, which confirms the uniqueness of the phytochrome system of gymnosperms [10].

In addition to the quality of light, plant growth is also affected by hormonal balance [11]. The lighting conditions cause the level of hormones in plants to change, which leads to a change in photosensitivity. For example, exogenous hormones can stimulate plant growth by acting as mediators in the processes of light signal transduction [12]. In turn, light by photoreceptors regulates the metabolism of various hormonal signals. Thus, PHYA affects the metabolic pathways of gibberellins and indoleacetic acid [11], as well as key components of light signaling, such as the transcriptional factors (TFs): PIF3, PIF4, and HY5 [13]. The main phytohormones are associated with light-mediated growth regulation [14–16], while the stimulation of cell growth involves auxins and cytokinins [17–19]. The light of different spectral compositions affects the activity of endogenous hormones through the regulation of secondary metabolism; for example, blue light promotes the accumulation of flavonoids, which in turn affects the polar transport of auxin [20].

The aim of this work was to understand how the conditions of light of different spectral composition affect the growth, morphometric, and photosynthetic characteristics of *Pinus sylvestris* seedlings. At the same time, special attention was given to the possible relationships of the processes of growth, photosynthesis, and respiration with the intensity of expression of the main genes encoding proteins of photosystems, and the genes involved in light and hormonal signalling of *Pinus sylvestris* plants under light of various spectral quality. The obtained results can be used to create artificial lighting systems in forest nurseries to accelerate the cultivation of planting material, and they also have applications in biotechnology.

#### **2. Materials and Methods**

#### *2.1. Plant Materials and Experimental Design*

Seeds of Scots pine (*P. sylvestris* L.) were collected in the Bryansk region (Bryansk, Russia) from high-productivity pine stands in complex forest types. Seeds were germinated and grown in hydroculture on individual substrates in polypropylene cartridges filled with 1% agar bungs in individual boxes of the climatic chamber under red (maxima of 660 nm), blue (maxima of 450 nm), red + blue (maxima of 660 and 450 nm) LEDs, as well as under fluorescent lamps (58 W/33–640, white fluorescent lamps (Philips, Pila, Poland), <sup>130</sup> ± <sup>10</sup> <sup>μ</sup>mol (photons) m−<sup>2</sup> <sup>s</sup><sup>−</sup>1) for 6 weeks. (Figure 1).

**Figure 1.** Emission spectra of the light sources used in the experiments. Spectra of blue light (BL) with a peak at 450 nm (1), blue + red (BRL) with two maxima at 445 nm and 660 nm (2), white fluorescent lamps (WFL) with a set of peaks in the visible spectral region (3), and red light (RL) with a peak at 660 nm (4).

After seed coat rupture and cotyledon expansion, the seedlings were transferred to a nutrient solution [21]. The seedlings were cultivated in6Lplastic trays (171 seed beds per tray) in a growth chamber that provided a constant air temperature of 24 ± 2 ◦C and a 16 h photoperiod. The nutrient solutions were constantly aerated and renewed once a week. During the week, a constant volume of the nutrient solution was maintained by adding distilled water.

The fresh biomass of the roots and needles was determined using an analytical balance (Scout Pro SPU123, Ohaus Corporation, Parsippany, NJ, USA) with an accuracy of 1 mg, after which the samples were fixed in liquid nitrogen and stored at −70 ◦C until biochemical analyses. The fixation was carried out under the conditions of the light in which the plants grew without exposure to another light.

#### *2.2. Pigment Contents*

The contents of chlorophyll *a* (Chl *a*) and *b* (Chl *b*) and total carotenoids (Car) in pigment extracts of all studied needles were determined spectrophotometrically in 80% acetone [22].

#### *2.3. Measurements of CO2 Gas Exchange*

The photosynthetic rate (Pn) was determined in a closed system under light conditions using an LCPro + portable infrared gas analyser from ADC BioScientific Ltd. (United Kingdom) connected to a leaf chamber. The CO2 uptake per leaf area (μmol m<sup>−</sup>2s−1) was determined. The rate of photosynthesis of the leaves in the second layer from the top was determined at a saturating light intensity of 1000 μmol (photons) m−<sup>2</sup> s−1. Previously, we recorded the light curves in the interval of intensities from 0 to 1200 μmol (photons) m−<sup>2</sup> s<sup>−</sup>1. The light intensities in the region from 600 to 1200 μmol (photons) m−<sup>2</sup> s−<sup>1</sup> were saturated. After measuring the rate of photosynthesis, the light was turned off, and the rate of dark respiration was measured.

#### *2.4. Determination of Photochemical Activity*

Fluorescence parameters characterizing the state of the photosynthetic apparatus were calculated on the basis of induction fluorescence curves obtained using data from the JIP test, which is usually used to evaluate the state of PSII. Chlorophyll (Chl) fluorescence induction curves (OJIP curves) were recorded with the setup Plant Efficiency Analyser (Handy-PEA, Hansatech Instruments Ltd., London, UK). For the JIP test, OJIP curves were measured under illumination with blue light at an intensity of 3500 μmol (photons) m−<sup>2</sup> s−<sup>1</sup> for 1 s.

On the basis of induction fluorescence curves (OJIP curves), the following parameters, which characterize the PSII photochemical activity, were calculated: Fv/Fm, the PSII maximum quantum photochemical yield, and PIABS, the PSII performance index [23,24]. Here, Fv is the value of variable fluorescence, equal to the difference between Fm and F0; F0 is the minimum amplitude of fluorescence (F), and Fm is the maximum amplitude of fluorescence. For calculation of the PIABS, the following formula was used:

PIABS = (Fv/Fm)/(M0/Vj) × (Fv/F0) × (1 − Vj)/Vj); M0 = 4 × (F300<sup>μ</sup><sup>s</sup> − F0)/(Fm − F0); and Vj = (F2ms − F0)/(Fm − F0)

where M0 is the average value of the initial slope of the relative variable fluorescence of Chl *a*, which reflects the closing rate of the PSII reaction centers, and Vj is the relative level of fluorescence in phase J after 2 ms.

PAM fluorimetry (Junior-PAM, Walz, Germany) was used to evaluate the photosynthetic apparatus state. The values F0, Fv, Fm, Fm , and F , as well as the PSII maximum (Fv/Fm) and effective Y(II) (Fm −Ft)/Fm photochemical quantum yields and nonphotochemical quenching (NPQ) (Fm/Fm −1), were determined. Here, Fm and Fm are the maximum Chl fluorescence levels under dark- and light-adapted conditions, respectively. Fv is the photoinduced change in fluorescence, and Ft is the level of fluorescence before a saturation impulse is applied. F0 is the initial Chl fluorescence level. Actinic light was switched on for 10 min [I = 125 μmol (photons) m−<sup>2</sup> s<sup>−</sup>1].

#### *2.5. RNA Extraction and qRT-PCR*

RNA isolation was performed according to the method of Kolosova et al. (2004) [25] with some modifications suggested by Pashkovskiy et al. (2019) [26]. The quantity and quality of the total RNA were determined using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). cDNA synthesis was performed using the M-MLV Reverse Transcriptase Kit (Fermentas, Canada) and the oligo (dT) 21 primer for genes of nuclear coding and random 6 (Evrogen, Moscow, Russia) for chloroplast coding genes expression. The expression patterns of the genes were assessed using the CFX96 Touch™ Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA). The transcript levels were normalized to the expression of the *Actin 1* gene. The mRNA levels were expressed as a ratio of the corresponding values for the WFL plants. The relative gene-expression signal intensity at the WFL plants was considered to have a value of 1. Gene-specific primers (Table 1) for photosystem II protein D1 (*psbA*, ABO77179.1), cryptochrome 1 (*Cry1*, K7R334), cryptochrome 2 (*Cry2*, T2FFB6), phytochrome P (*phyP*, AIY54822.1), phytochrome N (*phyN*, AFV79519.1), phytochrome O (*phyO*, A7Y6Q6), phytochrome-interacting factor 3 (*PIF3*, D5ABG4), chalcone synthase (*CHS*, AF543757.1), stilbene synthase (*STS*, S50350.1), histidine-containing phosphotransfer 1 (*HPT1*, ALN42232.1), type-A response regulators (*RR-A*, FJ717710.1), and auxin-induced protein 1 (*Aux/IAA*, AY289600.1) were selected using nucleotide sequences from the National Center for Biotechnology Information (NCBI) database (Available online: http://www.ncbi.nlm.nih.gov (accessed on 15 May 2021)) and database www.uniprot.org (Available online: http://www.uniprot.org (accessed on 20 May 2021)) with Vector NTI Suite 9 software (Invitrogen, Carlsbad, CA, USA).


**Table 1.** The primers for qRT-PCR analysis.

#### *2.6. Histochemical Studies of the Hypocotyls*

Anatomical studies were performed using light microscopy methods on live preparations of cross-sections of hypocotyls of *P. sylvestris* seedlings prepared immediately before the study. Sections 10–30 μm thick were prepared using an HM650 V vibrating blade microtome (Thermo Fisher Scientific, Waltham, MA, USA) through the central part of the organ. The obtained sections were stained with 3% phloroglucinol/HCl reagent (Sigma, P3502) for 2 min and then washed with phosphate buffered saline (70 mM, pH = 7.4) [27]. The sections were photographed under an Imager D1 light microscope (Carl Zeiss, Oberkochen, Germany) using a Levenhuk M800 PLUS digital photo attachment with a resolution of 8.0 MPI (Levenhuk, Tampa, FL, USA). The resulting images were processed with the public domain software ImageJ v.1.49 (NIH; http://rsb.info.nih.gov/ij). The diameter of the hypocotyl, the number of lignified xylem cells, the sectional area of the hypocotyl, the area of the xylem, and the average sectional area of one xylem cell were determined on the sections of the hypocotyls.

#### *2.7. Statistics*

The number of biological replicates for the determination of the fresh biomass of the roots and needles ranged from 27 to 39; 13 biological replicates were performed for the histochemical studies of the hypocotyls, and 6 biological replicates were used for photosynthetic and respiration rates as well as photochemical activity. Each plant sample fixed in liquid nitrogen was treated as a biological replicate; therefore, there were six biological replicates for the pigment contents and gene expression analyses.

The data were statistically analyzed using SigmaPlot 12.3 (Systat Software, San Jose, USA) with one-way analysis of variance (ANOVA) followed by Duncan's method for normally distributed data (in the figures, significant differences are denoted by different capital letters). Kruskal–Wallis one-way ANOVA was performed on ranks followed by a Student-Newman–Keuls post hoc test or by a Dunn's post hoc test for non-normally distributed data and data with unequal variance (in the figures, significant differences are denoted by different italic letters for the Student-Newman–Keuls post hoc test or by different boldface italic type for the Dunn's post hoc test). Different letters were used to indicate significance at *p* < 0.05. The values presented in the tables and figures are the arithmetic means ± standard errors.

#### **3. Results**

#### *3.1. Growth and Morphological Parameters*

Narrowband light caused marked changes in plant morphology and growth (Figure 2).

**Figure 2.** The morphological changes of *P. sylvestris* seedlings with dependence on light quality. WFL (**A**), BRL (**B**), BL (**C**), and RL (**D**).

Thus, RL increased the weight of the needles by 2.6 times, and BL increased the weight of the needles by 2.1 times relative to the WFL taken as a control (Figure 3A). BL caused hypocotyl elongation, and plants with RL formed short, larger plants. In addition, plant root weights in the RL variant were 3.8 times higher than the root weight in the control (WFL) and, on average, 2.7 times higher than in the other experimental variants

(Figure 3B). Moreover, there was an increase in both the fresh and dry weight of the root in RL (Figure 3B,E) and dry weight of needles in BL and RL in relation to WFL control (Figure 3D). In general, RL led to a more intensive development of the root system of the seedlings and an increase in the number of lateral roots of the first and second orders (Figure 2). Under the influence of RL, in the hypocotyls of the seedlings, 1.7 times more xylem cells were formed compared to the other variants of the experiment (Figure 3C), in the absence of any differences in the total area of the hypocotyls (Figure 3F). It is important to note that the growth of xylem under RL was not accompanied by thickening of the hypocotyls (Figure 3F), but the area of the xylem increased relative to other tissues (Figure 3G,H), while the diameter of xylem cells did not differ significantly (Figure 3I).

**Figure 3.** Effect of light quality on the needle (**A**) and root (**B**) fresh weight (mg); number of xylem cells (**C**) (pieces); needle (**D**) and root (**E**) dry weight (mg, DW); hypocotyl diameter (**F**) (mm); xylem cross-section area (**G**) (mm); xylem cross-section area and hypocotyls cross-section area ratio (**H**) (%); xylem cell cross-section area (**I**) (μm2); and content of Chl *a* (**J**) mg/g DW, Chl *b* (**K**) mg/g DW, and total carotenoids (**L**) mg/g DW in *P. sylvestris* seedlings. Values are the mean ± SE. Different capital

letters denote statistically significant differences in the means at *p* < 0.05 (ANOVA followed by Duncan's method). Different italic letters denote statistically significant differences in the means at *p* < 0.05 (Kruskal–Wallis ANOVA of the ranks followed by the Student-Newman-Keuls post hoc test). Different boldface italic letters denote statistically significant differences in the means at *p* < 0.05 (Kruskal–Wallis ANOVA of the ranks followed by the Dunn's method).

#### *3.2. Contents of Photosynthetic Pigments*

When grown on a narrowband RL and BL, a reduced content of chlorophylls and carotenoids was observed (by an average of 10–20%), while the BRL and WFL variants were comparable (Figure 3J–L).

#### *3.3. Fluorescence Parameters and CO2 Gas Exchange*

The photosystem II (PSII) maximum quantum yield (Fv/Fm) was approximately 0.81 and did not depend on the light quality used in the experiments (Table 2).

**Table 2.** Effect of light quality on the net photosynthetic and respiration rates, R/Pn ratio, (Pn–R) difference, PSII maximum quantum yield (Fv/Fm), effective quantum yield Y(II), performance index PSII (PIABS), and nonphotochemical fluorescence quenching (NPQ) in 6-week-old *P. sylvestris* seedlings. Values are the mean ± SE. Different normal-type letters denote statistically significant differences in the means at *p* < 0.05 (ANOVA followed by Duncan's method).


The PSII effective quantum yield (Y(II)) was the lowest in BRL plants (0.44); at the same time, in BL and WFL plants, this indicator remained at a comparable level and amounted to approximately 0.51. The highest value of the Y(II) parameter was observed when growing seedlings under RL (0.57) (Table 2). The non-photochemical quenching (NPQ) parameter was the smallest in RL plants. When plants were grown on BL and WFL, NPQ was comparable and amounted to approximately 0.90; the highest NPQ value (1.67) was observed in the BRL plants (Table 2).

The intensity of CO2 gas exchange did not differ noticeably between the samples. The largest value of the parameter Pn (16.3 μmol CO2 m−<sup>2</sup> s−1) was observed in the BRL variant, and the smallest value was 12.1 μmol CO2 m−<sup>2</sup> s−<sup>1</sup> in the RL variant, while the photosynthesis rate in the seedlings under the WFL and BL conditions was intermediate (Table 2).

The respiration rate (R) in the WFL and BRL variants was the highest (approximately 8.8 μmol CO2 m−<sup>2</sup> s−1), while in BL and RL plants, this value was reduced to 5.1 and 2.1 μmol CO2 m−<sup>2</sup> s−1, respectively. As a consequence, the calculated parameters of the R/Pn ratio and the Pn–R carbon balance changed accordingly. The respiration/photosynthesis ratio was the highest in the WFL and BRL variants, lower in the BL variant, and the lowest in the RL plants (0.17). At the same time, the highest value of the carbon balance, assessed by the difference between the rates of photosynthesis and respiration, was observed in the RL variant (Table 2).

The PSII performance index (PIABS) was on average 1.5 times higher in the RL variant than in the other variants. At the same time, all other options did not differ significantly among themselves (Table 2).

#### *3.4. Gene Expression*

The transcript level of genes involved in hormonal signaling of cytokinins and auxins significantly changed in the variants of narrow-band light. Expression of the *HPT1* gene in the needles of RL plants was increased almost two times, and in the roots, it was 2.7 times higher than that in the WFL control (Figure 4A,D).

**Figure 4.** Effect of light quality on the transcript levels of different groups of genes in *P. sylvestris* seedlings. *HPT1, RR-A*, and *Aux/IAA* in needles (**A**–**C**) and roots (**D**–**F**). The mRNA levels of the genes were expressed as the BRL, BL, RL/WFL ratio (fold change BRL, BL, RL/WFL). Values are the mean ± SE. Different capital letters denote statistically significant differences in the means at *p* < 0.05 (ANOVA followed by Duncan's method). Different italic letters denote statistically significant differences in the means at *p* < 0.05 (Kruskal–Wallis ANOVA of the ranks followed by the Student-Newman-Keuls post hoc test).

At the same time, the level of *RR-A* transcription in the RL variant was 3.3 and 3.0 times higher than that in WFL needles (Figure 4B) and roots (Figure 4E), respectively. In contrast, in the roots of the BL variant, *RR-A* expression was more than 2-fold lower than that in the WFL control (Figure 4E). In the needles of the seedlings, the expression of the *Aux/IAA* gene in the RL and BL plants was, on average, 2.8 times higher than that in the WFL control (Figure 4C). At the same time, in the roots, the level of *Aux/IAA* transcripts was higher only in the RL variant (Figure 4F).

We also studied the transcription of genes responsible for light signaling and the synthesis of secondary metabolites, such as the transcription factors *PIF3*, *CHS*, and *STS*. The expression level of the *PIF3* gene in the RL variant compared to that of WFL was reduced in needles by 30% (Figure 5A), while that of the *CHS* gene, on the contrary, increased 1.5 times compared to those of BRL and WFL (Figure 5B).

An increase in the transcript level of the *STS* gene was also observed more than 11 times in needles (Figure 5C) and more than 23 times in roots (Figure 5F) in the RL variant, and approximately 4 times in needles and 6 times in roots in the BL variant (Figure 5C,F).

Over the course of the experiment, the gene expression of the main proteins of photosystems I and II was studied. Among the large number of analyzed genes (*psbA,B,C,D,S; petA,C,D,E; psaA,B; flvA,B; Lhc1,2*), only the transcription levels of *psbA* encoding the PSII key protein D1 changed significantly and reliably. Thus, in variants BRL and BL, a twofold increase in the level of *psbA* transcripts was observed, while RL did not cause changes in the expression of this gene (Figure 6A).

**Figure 5.** Effect of light quality on the transcript levels of different groups of genes in *P. sylvestris* seedlings. *PIF3, CHS*, and *STS* in needles(**A**–**C**), and roots (**D**–**E**). The mRNA levels of the genes were expressed as the BRL, BL, RL/WFL ratio (fold change BRL, BL, RL/WFL). Different italic letters denote statistically significant differences in the means at *p* < 0.05 (Kruskal–Wallis ANOVA of the ranks followed by the Student-Newman–Keuls post hoc test).

**Figure 6.** Effect of light quality on the transcript levels of different groups of genes in *P. sylvestris* needles. *psbA* (**A**), *Cry1* (**B**), and *Cry2* (**C**); and *phyP* (**D**), *phyN* (**E**), and *phyO* (**F**). The mRNA levels of the genes were expressed as the BRL, BL, RL/WFL ratio (fold change BRL, BL, RL/WFL). Values are the mean ± SE. Different capital letters denote statistically significant differences in the means at *p* < 0.05 (ANOVA followed by Duncan's method). Different italic letters denote statistically significant differences in the means at *p* < 0.05 (Kruskal–Wallis ANOVA of the ranks followed by the Student-Newman–Keuls post hoc test).

In addition, the gene expression of apoproteins of the main blue and red light photoreceptors was studied (Figure 6B–D,F). Significant differences were observed in the *phyN* gene transcripts, the level of which was reduced by almost 2-fold in BL and RL relative to the WFL control (Figure 6E). Significant but negligible differences were observed in the transcript levels of *Cry1* and *Cry2* genes (Figure 6B,C).

#### **4. Discussion**

Seedlings of *P. sylvestris* are photophilous; however, in the first few seasons of their life, they can only be higher than the surrounding herbaceous plants for a short period of time, which causes a lack of light, primarily in the red and blue spectral ranges. It is known

that RL, in contrast to BL, has a significant effect on the stem growth, stem diameter, and size and dry weight of *Picea abies* needles [20]. Thus, in contrast to growing under BL and WFL, RL increased the biomass and stem diameter of *Brassica oleracea* plants [28]. At the same time, photosynthesis in RL plants was noticeably higher than in other variants, which probably led to an increasing biomass [28]. In our work, RL influenced the morphology of *P. sylvestris* seedlings, which was manifested in an increase in the mass of the root system by more than 3.8 times, complications of root branching, and an increase in the mass of the needles by 2.6 times in comparison with WFL. At the same time, RL caused a slight decrease in CO2 gas exchange, as well as a significant (more than four times) decrease in respiration intensity relative to those of the WFL control (Table 2). This indicates a greater assimilation of carbon in RL plants. The slight difference in the pigment content is consistent with the fact that their photosynthetic apparatus is not dependent on light.

Regarding photochemical processes, the effective PSII quantum yield Y(II) was the highest in RL plants but the value of NPQ was small. We suppose that the increased Y(II) and complementary decreased NPQ are due to preferential excitation of photosystem I (PSI) by RL with a wavelength >685 nm. The contribution of the long-wave light is quite significant in red LEDs spectrum (Figure 1). Such light can lead to faster re-oxidization of the plastoquinone pool and reopening of PSII reaction centers [29]. As a result, reaction centers use absorbed light more efficiently.

Gymnosperms have the ability to synthesize chlorophyll in the dark due to the presence of the three genes of light-independent protochlorophyllide oxidoreductase L, N, and B (*ChlL*, *ChlN*, and *ChlB*) involved in the light-independent reduction of protochlorophyllide to chlorophyllide [30]. This also confirms the presence of special light regulation in conifers, which makes them a unique object of research.

The features of development and metabolism in plants induced by light of different spectral composition are primarily mediated by changes in the expression of lightdependent genes [20,30], including those encoding chloroplast proteins, photoreceptor apoproteins, transcription factors, and enzymes involved in the biosynthesis of secondary metabolites [31] and phytohormone signaling. In our work, we showed that several genes involved in the response to RL or BL are expressed in different ways. For example, the level of transcription of the *psbA* gene of the PSII main protein D1 increased in BL and BRL plants but decreased in RL plants (Figure 6A). This response to RL, as well as the accumulation of *psbA* transcripts under BL and RL conditions, is a typical response for most flowering plants [32]. Unfortunately, we did not observe noticeable changes in the expression of photoreceptor genes, with the exception of *phyN* (Figure 6B–F); on the other hand, under conditions of prolonged exposure to light of different spectra, it is impossible to exclude the presence of a sufficient number of active forms of photoreceptors, as well as the presence of regulation at the level of light signaling. In another work, it was shown that *phyN* is able to respond to different ratios of BL and RL in Scots pine seedlings of different growing regions [6].

The quality of light influences photomorphogenesis, photosynthesis, and plant growth through appropriate photoreceptors [33,34]. However, no significant difference in the expression level of photoreceptor genes was found between the light variants BL and RL, which is consistent with previous studies performed on *A. thaliana* seedlings [35]. The gene expression profiles of *A. thaliana* plants grown under BRL, RL, and BL were similar in all variants, and a significant proportion of differentially expressed genes under BL were also induced under RL. This indicates that the expression of light-regulated genes in *P. sylvestris* is not a unique response to BL or RL and that light of different spectral composition is able to regulate metabolic patterns in a similar way through the regulation of light signaling genes.

Transcription factors play an important role in the regulation of photosynthetic apparatus sensitivity to light of different spectral composition, since they, together with photoreceptors, are involved in the transduction of both light and hormonal signals. Phytochrome signaling transcription factors (PIFs) are important negative regulatory proteins

that can alter the expression of a number of associated genes [6,36]. We observed a 30% decrease in the expression of the *PIF3* gene in RL plants, which may be associated with the activation of light signaling at the level of transcription of the corresponding genes (Figure 5A,D). These results demonstrate that the mechanisms by which light of different spectral composition controls the growth of *P. sylvestris* may involve angiosperm light signaling pathways. Transcription factors, together with photoreceptors, can influence the expression of hormonal signaling genes. Thus, a direct link between cytokinin signaling and light was found in a study demonstrating the key role of A-type RR regulators of cytokinin signaling [37–39]. RL induces *RR-A* expression in a PHYB-dependent manner. Thus, the overexpressing *RR-A* leads to hypersensitivity to RL [37]. Later research showed an important role for RR-A in photomorphogenesis [40,41]. RR-As are able to interact with PHYB via the cytokinin receptor (AHK). In support of this, we observed an increased expression of one of the main proteins of cytokinin signaling transduction (*HPT1*) by more than two times in roots and needles in the RL variant (Figure 4A,D). In addition, the PIFs are also involved in phytochrome-mediated regulation of auxin signaling under RL conditions, since these TFs are able to bind to the promoter regions of the *Aux/IAA* genes. PIFs modulate plant growth by directly controlling the expression of auxin signaling genes [11]. We assume that this, to a certain extent, explains the greater number of xylem cells in hypocotyls in the RL variant and, as a consequence, a greater accumulation of plant biomass (Figure 4C,F).

Along with the growth and development of plants, changes in the spectral composition of light also affect secondary metabolism. Chalcone synthase, the first enzyme in the biosynthesis of flavonoids, is expressed in needles of RL plants and in roots of BL plants (Figure 5B,E). In our study, RL stimulated gene families associated with the biosynthesis of the flavonoids (*CHS*, *STS*). Stilbenes are a family of polyphenolic secondary metabolites that act as phytoalexins [42,43]. Previously, it was shown that treatment with RL suspension culture of grape cells increased the biosynthesis of stilbenes [44,45]. In our study, we observed an increase in *STS* expression in roots and needles by more than 3.5 times in BL plants and more than 10 times in RL plants (Figure 5C,F). It can be assumed that, as in grape plants, stilbenes are involved in the photoadaptation of *P. sylvestris* plants to narrow-band RL and BL.

#### **5. Conclusions**

In this work, we tried to answer the question of what spectral range of light can be most favorable for the growth of *P. sylvestris* seedlings. It was shown that the RL spectral range is most favorable for growing *P. sylvestris* seedlings in a hydroculture, which manifested itself in both a greater mass of aboveground and underground organs and in an increase in the number of xylem cells. We found an increase in the level of transcripts of genes for auxin and cytokinin signaling (*HPT1, RR-A*, and *Aux/IAA*) and a decrease in the expression of TF *PIF3*, which in turn could activate the expression of a number of genes associated with the synthesis of secondary metabolites (*CHS, STS*). Based on the data obtained, we assumed that the large biomass of *P. sylvestris* plants under the RL might be due to a large accumulation of carbon in the needles, which corresponds to a better balance between photosynthesis and respiration, as well as to the increased activity of cytokinins and auxins in seedlings. Although *P. sylvestris* seedlings are able to grow in conditions of low RL content at the beginning of ontogenesis, better RL radiation can significantly improve their growth and development. The obtained results can serve as a basis for the development of a technology for the accelerated cultivation of planting material during reforestation when growing seedlings under artificial lighting, and they can also be used in biotechnology.

**Author Contributions:** Investigation, formal analysis, data curation, and responsible for the experimental part of the manuscript: P.P., V.D.K., Y.I., A.I., and A.K.; obtained main results: P.P., Y.I., A.I., A.S., V.S.; designed the experiments, interpreted the main results, drew the main conclusions, and

prepared the first version of the manuscript: V.D.K., P.P., S.I.A. and V.V.K. All authors have read and agreed to the published version of the manuscript.

**Funding:** The research was carried out within the state assignment of Ministry of Science and Higher Education of the Russian Federation (theme No. 121040800153-1).

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The data presented in this study are available on request from the corresponding authors. The data are not public.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


### *Article* **The Combined Effect of ZnO and CeO2 Nanoparticles on** *Pisum sativum* **L.: A Photosynthesis and Nutrients Uptake Study**

**Elzbieta Skiba ˙ 1,\* , Monika Pietrzak <sup>1</sup> , Sława Gli ´nska <sup>2</sup> and Wojciech M. Wolf <sup>1</sup>**


**Abstract:** Cerium oxide nanoparticles (CeO2 NPs) and zinc oxide nanoparticles (ZnO NPs) are emerging pollutants that are likely to occur in the contemporary environment. So far, their combined effects on terrestrial plants have not been thoroughly investigated. Obviously, this subject is a challenge for modern ecotoxicology. In this study, *Pisum sativum* L. plants were exposed to either CeO2 NPs or ZnO NPs alone, or mixtures of these nano-oxides (at two concentrations: 100 and 200 mg/L). The plants were cultivated in hydroponic system for twelve days. The combined effect of NPs was proved by 1D ANOVA augmented by Tukey's post hoc test at *p* = 0.95. It affected all major plant growth and photosynthesis parameters. Additionally, HR-CS AAS and ICP-OES were used to determine concentrations of Cu, Mn, Fe, Mg, Ca, K, Zn, and Ce in roots and shoots. Treatment of the pea plants with the NPs, either alone or in combination affected the homeostasis of these metals in the plants. CeO2 NPs stimulated the photosynthesis rate, while ZnO NPs prompted stomatal and biochemical limitations. In the mixed ZnO and CeO2 treatments, the latter effects were decreased by CeO2 NPs. These results indicate that free radicals scavenging properties of CeO2 NPs mitigate the toxicity symptoms induced in the plants by ZnO NPs.

**Keywords:** combined effect; *Pisum sativum* L.; nanoparticles; cerium oxide; zinc oxide; metal uptake; photosynthesis; hydroponic culture

#### **1. Introduction**

Nowadays, synthetic nanomaterials (NMs) are finding applications in almost all material aspects of human life and are particularly vital for contemporary technology and medicine [1–5]. The most important are sustainable energy production and storage, electronic devices, catalysts, sensors and adhesives, high-quality petro- and agrochemicals [6–14]. Their widespread use raises fundamental questions related to the environment, pollution, and safety [15–17].

In the diverse world of nanomaterials metal oxides firmly occupy a unique position. Their global market in 2020 was worth almost USD 5.3 billion with several forecasts indicating its steady rise to USD 9.3 billion in 2027 [18]. Obviously, this growing stream of NMs cannot be completely isolated from soil, air, or water and finally will find its way to terrestrial living organisms. Therefore, complex interactions of nanoparticles (NPs) with the biota and their further environmental fate deserve additional studies.

In a number of comprehensive papers on interactions of nanoparticles with living organisms, the authors investigated the role of chemical composition, particle shape, size, and mechanisms of aggregation of NPs [19–22]. The beneficial or harmful effects of NPs were also examined. Owing to their unique properties, nanomaterials can be successfully used in fertilizers, pesticides, or as dedicated chemical carriers or sensors [23–25]. This increasing flux of diverse nanomaterials approaching soil and plant environments should not be left without comprehensive studies on their migrations, uptake, and toxicities.

**Citation:** Skiba, E.; Pietrzak, M.; Gli ´nska, S.; Wolf, W.M. The Combined Effect of ZnO and CeO2 Nanoparticles on *Pisum sativum* L.: A Photosynthesis and Nutrients Uptake Study. *Cells* **2021**, *10*, 3105. https:// doi.org/10.3390/cells10113105

Academic Editors: Suleyman Allakhverdiev, Alexander G. Ivanov and Marian Brestic

Received: 20 September 2021 Accepted: 5 November 2021 Published: 10 November 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

The emerging picture suggests dynamic processes which act in complicated matrices. Identification of interactions among various substances poses a real challenge.

Obviously, nanoparticles affect plant metabolism in a number of ways and finally introduce changes to plant physiology at cellular, organ, and individual plant levels [26–29]. In particular, photosynthesis—the essential, bioenergy-generating process [30]—may be either facilitated or hampered by nanoparticles [31–36]. As pointed out by Du et al. [37] and Tighe-Neira et al. [35], the latter may be conveniently examined by gas exchange parameters augmented with the contents of photosynthetic pigments. Unfortunately, results are rarely completely consistent and a wide range of plant responses to nanoparticles could be expected. Notably, metal oxide NPs usually alter the photosynthesis rate, photochemical fluorescence, and quantum yield in plants [37] with ZnO and CeO2 being among the most active. Mukherjee et al. [38] described the negative effect of ZnO NPs at soil concentrations 125–500 mg/L on chlorophyll activity in *Pisum sativum* L. It results from the substitution of the Mg atoms at chlorophyll centers by Zn which finally hampers the photosynthesis process. The opposite effect was observed by Reddy Pullagurala et al. [39] during cilantro (*Coriandrum sativum*) cultivation. Supplementation with 100 or 200 mg/L ZnO NPs induced photosynthetic pigments production and boosted photosynthesis.

The ambient character of nanoparticulate cerium oxide combined with its increasing abundance in the environment makes this substance a useful agent to study plant metabolism effects triggered by anthropogenic nanomaterials. In particular, Wu et al. [32] demonstrated that CeO2 NPs (nanoceria) are a reactive oxygen species (ROS) scavenger in leaf mesophyll cells and defend the chloroplast photosynthetic machinery from abiotic stresses. On the other hand, the negative effect of nanoceria on photosynthesis in soybean plants was investigated by Li et al. [40]. They pointed out several aspects which are at stake, namely: inhibited conversion efficiency of C5 to C3 in the Calvin–Benson cycle, destruction of thylakoid membranes, and reduced chlorophyll synthesis and activity. All these factors participate in the final plant destruction.

Regrettably, the most relevant works on the subject are aimed at particular types of nanoparticles. Investigations of combined, mutual effects induced by either mixture of NPs or their additives and stabilizers are quite scarce. In real ecosystems, nanoparticles rarely play a solo performance, which is especially important in modern efficient agriculture. Increasing pressure on massive food production for the rapidly growing population has prompted the development of new, smart, and efficient agrochemicals [41–44].

The fertilizing effect of CeO2 and ZnO NPs attracted the attention of several research groups. It is quite well documented that cerium oxide NPs can alleviate plant salinity stress, act as a catalyst in chlorophyll production, and in scavenging reactive oxygen species which stabilize the chloroplast structure and cell walls [45–47]. On the other hand, nanoparticulate zinc oxide may be used to counteract cadmium toxicity in wheat and elevate zinc concentrations in plants. Thus, it can be a useful agent for Zn biofortification in cereals plantations and help to overcome the well recognized hidden hunger in humans resulting from the deficiency of Zn in cereals [25]. In this respect, optimization of photosynthetic efficiency by nanoparticles is a highly promising approach for a smart increase of crop production [48].

Mixtures of nanoparticles that are present in the growth environment affect plant development in a different way than single species [49–51]. In particular, the combined, mutual interactions between ZnO and CeO2 are quite likely indeed. Both substances often coexist in all compartments of the environment and their simultaneous presence in the environment is more than likely. Our previous works on *Pisum sativum* L. were related to metal migration strategies as induced by single stressors such as CeO2 or ZnO nanoparticles [52,53]. We have shown that zinc species alter Cu, Mn, and Fe uptake and their further migration in green pea. On the other hand, low concentrations of cerium oxide NPs increased the photosynthesis rate. Those investigations had a model character and did not account for combined effects as triggered by ZnO and CeO2 together in real plant matrices. This study enhances this approach significantly. Here, we examine the mixture of

nanometric cerium and zinc oxides. The methodology has been based on hydroponic pot experiments [54]. Pea is frequently applied in system biology experiments and is treated as a non-model plant with a roughly complete genome structure [55,56]. It is cultivated worldwide [57] and additive interactions which affect nutrient uptake and enhance plant growth yield are of practical relevance.

#### **2. Materials and Methods**

#### *2.1. Nanoparticles Characteristic*

CeO2 NPs and ZnO NPs were purchased from the Byk (Byk-Chemie GmbH, Wesel, Germany) as commercially available products. The properties of nanoparticles, including average particle size, transmission electron microscopy (TEM) images, and zeta potential are given in Table S1 [52,54].

#### *2.2. Plants Growth Conditions and Treatments*

*Pisum sativum* L. plants were cultivated under hydroponic conditions in Hoagland's nutrient solution. The composition of the growing medium and the detailed setup of the experiment were described previously [53,54]. The seeds, ("Iłówiecki" sugar pea, "PNOS" Co., Ltd., Ozar ˙ ów Mazowiecki, Poland), sterilized in 70% ethanol were germinated in dark for 3 days. Next, the seedlings (at BBCH 09 phenological stage [58]) were transferred to perforated plastic plates and placed in containers with the nutrient solution. Each growing vessel contained 26 pea seedlings. *Pisum sativum* L. was grown in Hoagland's nutrient solution for 4 days in a controlled environment: light of 170 μmol/m2 intensity, average temperature 21 ± 3 ◦C, and 16/8 h day/night photoperiod. On the 5th day, the nutrient solutions were supplemented with nanoparticles. The treatments were as follows, CeO2 NPs: 100 mg (Ce)/L; CeO2 NPs: 200 mg (Ce)/L; ZnO NPs: 100 mg (Zn)/L; ZnO NPs: 200 mg (Zn)/L; two mixtures of CeO2 and ZnO NPs: 100 mg (Ce)/L + 100 mg (Zn)/L and 200 mg (Ce)/L + 200 mg (Zn)/L. Plants grown in Hoagland's nutrient solution served as the control group. In each variant of the experiment, six growing vessels were used. Fresh liquid media were supplied every 48 h. *Pisum sativum* L. was harvested after 12 days of exposure when plants reached the BBCH 15 phenological stage.

#### *2.3. Growth Parameters*

Root and stem lengths were measured at the end of cultivation (Figure 1). Next, the roots were thoroughly rinsed with deionized water and separated from the shoots. The fresh weight of roots and shoots was measured and calculated per single plant (mg/plant). Prior to the chemical analysis, the collected fresh plants material was dried to a constant weight at 55 ◦C.

#### *2.4. Elements Content*

The dried samples of roots and shoots were digested in the mixture of concentrated HNO3 and HCl (6:1, *v*/*v*) using a Multiwave 3000 Anton Paar microwave reaction system (Anton Paar GmbH, Graz, Austria). The concentrations of Cu, Mn, Zn, Fe, and Mg were determined by a High-Resolution Continuum Source Atomic Absorption spectrometer HR-CS AAS (contrAA300, Analytik Jena, Jena, Germany). Additionally, the digested solutions were analyzed for Ce, Ca, and K concentrations by an Inductively Coupled Plasma–Optical Emission spectrometer ICP-OES (PlasmaQuant PQ 9000, Analytik Jena, Jena, Germany). Certified reference material of plant leaves of Oriental Basma Tobacco Leaves (INCT-OBTL-5) obtained from the Institute of Nuclear Chemistry and Technology, Warsaw [59] was used to check the reliability of the applied analytical procedures. Recoveries ranging from 96% to 108% were obtained. The detailed numerical data are given in Table S2.

**Figure 1.** The influence of nanoparticulate CeO2 and ZnO on: (**a**) root and stem length and (**b**) fresh weight of root and shoot as determined for a single pea plant. Concentrations of nanoparticles are given in mg/L of elemental cerium or zinc, the cultivation time was 12 days. Roots are represented by grey while above-ground parts are in green. Vertical bars represent standard deviations (*n* = 6). Distinct letters show the statistically significant differences among treatments as calculated with Tukey's HSD post hoc test. The probability level *p* = 0.95 was applied.

#### *2.5. Tolerance Index (TI) and Translocation Factor (TF)*

Tolerance indices (TI) and translocation factors (TF) were calculated for plants treated with nanoparticulate oxides. TI is defined as the ratio between the root length of treated plants and that of plants in the control group [60,61]. The effectiveness of metal translocation from roots to shoots was assessed using TF, defined as the ratio of average element content in shoots to that in roots [62,63].

#### *2.6. Photosynthetic Pigments*

Contents of photosynthetic pigments, i.e., chlorophyll a and b and carotenoids in *Pisum sativum* L. leaves were determined following the method developed by Oren et al. [64]. All pigments were extracted from leaves with 80% acetone and kept in the dark at 4 ◦C. The absorbance of extracts was measured at wavelengths 470, 646, and 663 nm by SpectraMaxi3x (Molecular Devices, San Jose, CA, USA) on samples centrifugated at 4000 rpm for 10 min. Contents of pigments were calculated by the formula proposed by Lichtenthaler and Wellburn [65]. For comparison, the nondestructive measurements of relative chlorophyll

content in fully expanded leaves at the fourth node were performed by the Soil Plant Analysis Development SPAD-502Plus chlorophyll meter (Konica–Minolta, Inc., Osaka, Japan).

#### *2.7. Photosynthetic Parameters*

Photosynthetic parameters, such as: leaf net photosynthesis (A), sub-stomatal CO2 concentration (Ci), transpiration (E), stomatal conductance (gs), photosynthetic water use efficiency (WUE), and photosynthetic CO2 response curve (A/Ci) were determined with a portable gas exchange analyzer (CIRAS-3; PP Systems, Amesbury, MA, USA). Measurements were performed 12 days after the administration of nanoparticles on fully expanded leaves at the fourth node. PARi levels at 1000 μmol/m2s were obtained from an LED Light Unit (RGBW) connected to the gas analyzer. The temperature within the chamber was kept at 25 ◦C, relative humidity at 80%, and reference CO2 concentration at 390 μmol/mol. Photosynthetic CO2 response curves were collected at a CO2 concentration gradient ranging from 0 to 1500 μmol/mol. All measurements were performed on fully expanded leaves at the fourth node. The acclimatization time between measurements was 120 s. The results from each CO2 level were recorded three times. Two biochemical parameters: maximum carboxylation rate (Vcmax) and maximum electron transport rate (*J*max) were calculated in Rstudio (v.3.4.2; R Foundation for Statistical Computing, Vienna, Austria) [66] using the "plantecophys" package developed by Duursma [67].

#### *2.8. Statistical Analysis*

All treatments were replicated six times, numerical results are accompanied by their ± SD (standard deviation). Statistical analysis was performed with the OriginPro 2016 (OriginLab Corporation, Northampton, MA, USA) software. The normality of the sample distributions was proved by the Shapiro–Wilk test [68]. The initial hypothesis on equal variances of investigated populations was validated with the Bartlett test [69]. A one-way ANOVA with Tukey's post hoc approach was applied to validate differences between means. The probability level *p* = 0.95 was applied.

#### **3. Results and Discussion**

#### *3.1. Growth Parameters*

Several plant growth parameters were determined after twelve days of exposure time to illustrate the pea plant behavior under the combined treatments of nanoparticulate CeO2 and ZnO. Roots and stem lengths and their fresh weights are shown in Figure 1.

Almost all supplementations decreased roots length of pea as compared to the control treatment. Nevertheless, this effect is less pronounced for plants cultivated with the addition of ZnO (100 and 200 mg/L) than for the CeO2 (100 and 200 mg/L) treatments. It is directly correlated with the nanoparticles concentration applied. Notably, supplementation with mixed NPs resulted in roots shortening similar to that observed for relevant sole nanoceria concentrations. The order of tolerance indices (TI) supports above observations: (Zn 100) ≈ (Zn 200) > (Ce 100 + Zn 100) ≈ (Ce 100) > (Ce 200 + Zn 200) ≈ (Ce 200). Numbers in brackets represent supplementations of nanoparticles given in mg/L of elemental cerium or zinc, numerical values are presented in Table S3. Stem elongation was triggered by (Ce 100) and (Ce 200). A reverse effect was observed for (Zn 200) and (Ce 200 + Zn 200) administrations. Notably, the majority of treatments left the root mass unchanged with (Ce 100) and (Ce 200 + Zn 200) being the chief exceptions (Figure 1b). The biomass of the above-ground parts decreased upon all supplementations. The only exception was (Ce 100) with a biomass similar to that of a control sample.

#### *3.2. Cerium and Zinc Concentrations*

Cerium and zinc concentrations in roots and shoots along with their translocation factors are summarized in Table 1. The highest cerium content was determined for (Ce 200) treatment while the lowest levels were observed for combined (Ce 100 + Zn 100) and

(Ce 200 + Zn 200) administrations. Respective translocation factors were remarkably low, which indicates that plants accumulate cerium primarily in roots. Cerium was not detected in plants grown in Hoagland's nutrient solutions which were not supplemented with this element. Zinc is an essential element necessary for proper plant development [70,71]. The amounts of zinc accumulated by pea cultivated in solutions supplemented with the ZnO NPs alone were substantially larger than those observed for control samples. Combined treatments, namely (Ce 100 + Zn 100) and (Ce 200 + Zn 200) decreased Zn levels in both roots and shoots as compared to respective sole ZnO NPs administrations. However, all relevant concentrations highly exceeded critical toxic zinc level 300 mg/kg as suggested by Broadley et al. [70,71]. Similar to nanoceria administrations, the vast majority of zinc was immobilized in roots.

**Table 1.** Zinc and cerium concentrations in root and shoot of *Pisum sativum* L. grown under the sole or combined CeO2 and ZnO NPs treatments accompanied by translocation factors (TF). Concentrations of nanoparticles are given in mg/L of elemental cerium or zinc, respectively. The cultivation time was 12 days. Data are the means ± SD (*n* = 6). Distinct letters show the statistically significant differences among treatments as calculated with Tukey's HSD post hoc test (*p* = 0.95). nd: concentration below the detection limit (18 μg/L).


#### *3.3. Photosynthetic Pigments*

Measurements of photosynthetic pigments provide useful information on the physiological status of plants [72]. Concentrations of chlorophylls and carotenoids which are involved in the absorption and further transfer of light energy are prone to changes induced by inorganic chemical stressors [73]. Contents of chlorophyll a (Chl a), chlorophyll b (Chl b), and carotenoids (Car) in leaves of pea treated with nano-oxides are summarized in Figure 2a.

Significant differences among those parameters were observed. Almost all treatments prompted a decrease in photosynthetic pigments. The highest reduction was observed under (Zn 200) administration. Similar results were obtained by Hu et al. [74] who found that the lowest level of photosynthetic pigments in *Salvinia natans* grown in hydroponic conditions was reached in cultures supplemented with nanometric ZnO at 50 mg/L. Pea plants exposed to (Zn 100) and (Zn 200) showed initial symptoms of leaf chlorosis (Figure 2b). The chlorophyll loss associated with chlorosis is one of the typical symptoms of Zn excess in plants [75–77]. In this study, Zn levels in pea shoots under (Zn 100) and (Zn 200) administrations (Table 1) were significantly higher than the critical Zn toxicity level (>300 mg/kg) as quoted in the highly respected Marschner's Mineral Nutrition of Higher Plants [71].

Moreover, the lowest Chl a, Chl b, and Car concentrations were observed for the sole (Zn 200) treatment. Acute effects of nanoparticulate (Zn 200) were reduced by the (Ce 200) addition. It prompted the mutual, combined interactions which elevated photosynthetic pigments levels and significantly relieved leaf chlorosis symptoms. Following Wang et al. [75] and Broadley et al. [70], Zn may induce chlorosis of leaves by stimulating Mg, Fe, and Mn deficiency. These elements are crucial in the synthesis and stability of chlorophyll. A decline of green photosynthetic pigments in plants under ZnO NPs treatment was also reported by Zoufan et al. [78] who explained it by peroxidation of the chloroplast

membrane due to exacerbation of oxidative stress. Oxidative damage triggered by ZnO NPs was also reported by Salehi et al. [79]. In turn, CeO2 NPs have the ability to quench ROS, mainly produced in chloroplasts [80–82], and presumably mitigate stress induced by nanoparticulate ZnO.

**Figure 2.** The influence of nanoparticulate CeO2 and ZnO on: (**a**) photosynthetic pigments: chlorophyll a (Chl a), chlorophyll b (Chl b), and carotenoids (Car) in *Pisum sativum* L. The pigments were extracted from mature leaves after 12 days of contact with a particular nano-oxide. Vertical bars represent standard deviations (*n* = 6). Treatments with the same letter are not significantly different according to Tukey's post hoc test (*p* = 0.95) (**b**) appearance of pea leaves from each treatment.

Apart from the conventional wet chemical methods based on extraction of the chlorophyll pigments and their further spectrophotometric determination, the nondestructive measurements using the Soil Plant Analysis Development SPAD-502Plus chlorophyll meter were also performed (Figure 3). The results obtained by all those methods are highly correlated. The reduction in chlorophyll content expressed in SPAD units in *Pisum sativum* L., cultivated in soil supplemented by nanoparticulate ZnO, was also observed by Mukherjee et al. [38], and following Küpper et al. [83] was attributed to the Mg substitution by Zn.

**Figure 3.** The influence of nanoparticulate CeO2 and ZnO on chlorophyll content in *Pisum sativum* L. expressed in SPAD units. All measurements were performed on mature leaves on the 12th day from administration of the nanoparticles. Vertical bars represent standard deviations (*n* = 10). Treatments with the same letter are not significantly different according to Tukey's post hoc test (*p* = 0.95).

#### *3.4. Photosynthesis Parameters*

Photosynthesis efficiency is usually assessed with the gas exchange analysis of plant leaves [84–86]. Leaf net photosynthesis (A), transpiration rate (E), stomatal conductance (gs), sub-stomatal CO2 concentration (Ci), and water use efficiency (WUE) determined under either sole or combined nanoparticles treatments are collected in Figure 4. Net photosynthesis was encouraged by CeO2 supplementations and significantly reduced by ZnO additions, Figure 4a. The latter follows reduced chlorophyll content as induced by elevated zinc concentrations [75,87]. This effect is partially released by the nanoceria addition in combined (Ce 100 + Zn 100) and (Ce 200 + Zn 200) treatments. Zinc is an essential metal necessary for proper plant development [70]. However, above certain concentrations, it affects chlorophyll synthesis and photosystem II efficiency [88,89]. Transpiration and stomatal conductance behave in a similar way with the highest values observed for (Ce 100) and (Ce 200) treatments and their substantial decline induced by either (Zn 100) and (Zn 200) supplementations (Figure 4b,c). The sub-stomatal CO2 concentrations exhibit a more uniform pattern and are significantly less prone to ZnO supplementations (Figure 4d). The largest water use efficiency was observed for (Ce 100) and (Zn 100) treatments, Figure 4e. High concentrations of (Ce 200) and (Zn 200) nanoparticles prompted a decrease in WUE. Maximum rates of ribulose 1,5-bisphosphate (RuBP) carboxylation as catalyzed by Rubisco (Vcmax), electron transfer driving the regeneration of RuBP (*J*max), and CO2 compensation points for pea plants cultivated in Hoagland's solution supplemented with either sole or combined CeO2 and ZnO NPs are in Table 2 and Supplementary Figure S1. The highest Vcmax values were observed for the sole nanoceria augmentations [(Ce 100) and (Ce 200)] while the strongest decreases were induced by the sole (Zn 100) and (Zn 200) NPs additions. The electron transfer rates *J*max followed patterns observed for the RuBP regeneration. The lowest carbon dioxide compensation point was determined for the (Ce 100) treatment, the largest was obtained for (Zn 200).

Nanoceria is an effective free radicals scavenger and mimics the activity of several enzymes, namely superoxide dismutase (SOD), catalase (CAT), and oxidase (OXD) [32,90–92]. In this respect, it was classified by Korsvik et al. [93] as the first antioxidant nanoenzyme [90]. Stimulation of photosynthesis under abiotic stress mitigated by nanoceria in

*Arabidopsis thaliana* was investigated by Wu et al. [32,94]. They pointed out that nanoparticulate CeO2 reduced the stress induced by reactive oxygen species, namely •OH, which are not tackled by the usual plant enzymatic scavenging pathways. Those NPs had entered chloroplasts through the nonendocytic pathways, reduced the ROS concentrations, and finally increased the quantum yield of photosystem II, carbon assimilation rates, and chlorophyll content. The latter processes are governed by the reversible redox reactions between the Ce4+ and Ce3+ species which are followed by the oxygen vacancy generation or elimination [95–97]. Moreover, Cao et al. [22,98] identified a strong correlation between photosynthesis parameters and the applied doses of CeO2 NPs in soybean. The resulting photosynthesis enhancement was explained by the elevated Rubisco activity (Vcmax) and promotion of the NADPH regeneration rate which prompted the RuBP synthesis.

**Figure 4.** Leaf net photosynthesis A (**a**), transpiration E (**b**), stomatal conductance gs (**c**), sub-stomatal CO2 concentration Ci (**d**), and water use efficiency (WUE) (**e**) for *Pisum sativum* L. grown under the sole or combined CeO2 and ZnO NPs treatments. Concentrations of nanoparticles are given in mg/L of elemental cerium or zinc, respectively. The cultivation time was 12 days. Vertical bars represent standard deviations (*n* = 6). Distinct letters show the statistically significant differences among treatments as calculated with Tukey's HSD post hoc test (*p* = 0.95).



Nanometric ZnO affected the CO2 assimilation process in a roughly opposite way than CeO2 NPs. At low concentrations, zinc is a micronutrient necessary for proper plant development. At elevated levels (above 300 μg/g, plant dry weight), zinc becomes a toxic pollutant responsible for the generation of ROS [99,100]. It alters stomata morphology and formation. The decreased gs and E indicated an increased stomatal closure and restriction of the transpiration rate. Carbon dioxide enters pea leaves by diffusion through stomatal pores. The major photosynthesis limitations result either from diffusion-controlled restrictions in CO2 supply to the carboxylation sites or reduction in its consumption through the mitigated Rubisco activity and RuBP regeneration [101]. The decrease in the net photosynthesis A was correlated with the intercellular CO2 concentration Ci and accompanied by the simultaneous reduction in Vcmax and *J*max. These results suggest that ZnO NPs induced either stomatal or biochemical photosynthesis limitations. In the mixed ZnO and CeO2 treatments, the latter effects are attenuated by the free radical scavenging properties of nanoceria.

#### *3.5. Elements Content*

Copper, manganese, iron, magnesium, calcium, and potassium contents were determined in plants from all treatments. The results are presented in Figure 5 and Table S4. Roots and shoots were treated separately. A one-way ANOVA was initially used to evaluate concentrations of macro- and micronutrients in pea plants. The 0.95 probability level was applied. Both nano-oxides altered the uptake of elements and their further translocation to the green parts of pea (Table S5).

Nanoceria in both tested concentrations [(Ce 100) and (Ce 200)] significantly reduced the uptake of all three investigated heavy metals (Cu, Mn, and Fe) by the roots. On the other hand, nanoparticulate ZnO [(Zn 100) and (Zn 200)] behaved in a less obvious way. Roots uptake was increased for copper and decreased for manganese. The uptake of iron by the roots was not affected by (Zn 100) and was reduced by high zinc supplementation (Zn 200). The combined treatments, either (Ce 100 + Zn 100) or (Ce 200 + Zn 200), prompted more complex root responses. For Cu, the resulting uptakes were between those induced by the administration of either CeO2 or ZnO alone. The manganese levels were as low as those observed for the sole (Zn 100) and (Zn 200) supplementations. In the combined (Ce 200 + Zn 200) treatment Fe root level was as high as that in the control sample. However, (Ce 100 + Zn 100) treatment prompted much lower Fe concentrations. In green parts of the pea plants, the levels of Cu, Mn, and Fe were lower than those in roots. The only exception was manganese whose concentrations in shoots were higher for all samples supplemented with nanometric ZnO. Zinc hampers manganese uptake by roots. The latter element is essential for the water-splitting process during the light-dependent phase of photosynthesis. These circumstances facilitate Mn migration from roots to shoots. A quite similar situation was observed for magnesium and calcium. The former is an important chlorophyll cofactor while the latter is a structural component of photosystem II. Both ions are crucial for the overall photosynthesis yield. The additions of (Ce 100) and (Ce 200) NPs

alone decreased potassium levels in roots and shoots. Both were inversely proportional to the Ce supplementations. The nanometric ZnO in either sole or combined administrations further restrained potassium contents in pea roots. However, unlike in the case of the nanoceria additions, higher K levels were observed in shoots.

**Figure 5.** Copper (**a**), manganese (**b**), iron (**c**), magnesium (**d**), calcium (**e**), and potassium (**f**) concentrations in roots and shoots of *Pisum sativum* L. grown under the sole or combined CeO2 and ZnO NPs treatment. Concentrations of nanoparticles are given in mg/L of elemental cerium and zinc, respectively. The cultivation time was 12 days. Vertical bars represent standard deviations (*n* = 6). Distinct letters show the statistically significant differences among treatments as calculated with Tukey's HSD post hoc test (*p* = 0.95).

#### **4. Conclusions**

Nanomaterials alter plant metabolism in a number of ways with photosystems I and II being their important targets. This paper continues our investigations on plant

metabolism and uptake of nutrients in a model hydroponic environment subjected to nanoparticles pollution. Steadily increasing usage of nanomaterials in smart agriculture prompts thorough studies on their interactions with plant organisms. Nanoparticles rarely exist in the environment alone and their combined interactions can hardly be neglected.

Regrettably, relevant studies on their impact on plant photosynthesis are scarce. In this study, pea plants were cultivated in Hoagland's solutions supplemented with either sole or mixed cerium and zinc nano-oxides at 100 mg/L or 200 mg/L Ce or Zn levels. Despite relatively high sole CeO2 administration (200 mg/L), no morphological symptoms of phytotoxicity were detected in *Pisum sativum* L. Leaf net photosynthesis, water use efficiency, and fresh biomass production were stimulated at the 100 mg/L Ce concentration and only slightly suppressed at its higher level. Contrarily, ZnO NPs applied alone caused serious impairment of metal homeostasis, decreased the level of photosynthetic pigments, induced leaf chlorosis, and finally hampered photosynthetic efficiency. We proved that ZnO NPs induced stomatal and biochemical limitations of photosynthesis. Such dysfunctions could lead to the overproduction of ROS in chloroplast and induce oxidative stress. In mixed CeO2–ZnO NPs treatments, the beneficial effect of nanoceria was observed. In particular, pigments concentrations, leaf net photosynthesis, and maximum electron transport rate (*J*max) depressed by ZnO NPs were significantly alleviated when CeO2 NPs were present in the growing medium. It is well recognized that nanoceria has the potential to quench ROS. Therefore, we conclude that CeO2 NPs moderate ZnO NPs toxicity by protecting the photosynthetic apparatus in *Pisum sativum* leaves from oxidative stress trigged by Zn. Additionally, we observed that both nano-oxides affected the nutrients uptake and transport at all concentrations applied.

Reactive oxygen species are by-products of aerobic metabolic processes in plants [102,103]. They usually increase membrane permeability and initiate stress signals often leading to programmed cell death [104]. At certain concentrations, the presence of NPs in growing media elevates ROS levels and induces oxidative damage in plant species. On the other hand, plant organisms have developed advanced antioxidant systems which involve either enzymatic or non-enzymatic pathways stabilizing ROS levels [105]. Those systems are enhanced under exposure to NPs, perhaps as an adaptive response to alleviate stress. We speculate that either CeO2 or ZnO nanoparticles trigger oxidative stress in pea but only cerium dioxide acts as an antioxidant and reduces the stress symptoms while zinc oxide is mainly a prooxidant.

Our future studies will be aimed at the binary activity of nanoceria in agricultural plants. Namely, at high concentrations, it is a plant stressor that triggers ROS production while at certain, low levels nanoceria exhibits a ROS scavenging ability and supports the plant's defense mechanisms. The latter effect may find applications in agriculture and deserves further investigation.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/article/10 .3390/cells10113105/s1, Table S1: Characterization of CeO2 NPs and ZnO NPs, Table S2: Elements content in certified reference materials Oriental Basma Tobacco Leaves—INCT-OBTL-5 (*p* = 0.95, *n* = 8), Table S3: Root length and tolerance index (TI) of *Pisum sativum* L. treated with nanoparticulate CeO2 and ZnO, Table S4: ANOVA parameters for elements concentrations in roots and shoots of *Pisum sativum* L. plants across all treatments., Table S5: Translocation factors (TF) of nutrients from root to shoot in *Pisum sativum* L. plants grown under the sole or combined CeO2 and ZnO NPs treatments, Figure S1: Comparison of CO2 response curves (A/Ci) between control and treated plants.

**Author Contributions:** Conceptualization, E.S. and W.M.W.; methodology, E.S., M.P. and S.G.; validation, E.S. and W.M.W.; formal analysis, E.S. and M.P.; investigation, E.S. and M.P.; writing original draft preparation, E.S., M.P. and W.M.W.; writing—review and editing, E.S., M.P., S.G. and W.M.W.; visualization, E.S. and M.P.; supervision, E.S. and W.M.W. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work received support from the Regional Fund for Environmental Protection and Water Management in Lodz, Poland (Grant Number: 58/BN/D2018); additional funding from the Institute of General and Ecological Chemistry of Lodz University of Technology is also acknowledged.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The data presented in this study are available on request from the corresponding author.

**Acknowledgments:** The authors wish to thank Lilianna Ch ˛eci ´nska for consultations on the solid structures of nano-oxides and their influence on properties of nanomaterials in micellar forms. Jakub Kubicki is kindly acknowledged for his support in heavy metals determination; Sylwia Michlewska and Sławomir Kadłubowski for their help in zeta potential measurements. The European University Foundation is acknowledged for advising on the legal and social dimensions of this study.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

