Seeds of Vicia lens (L.) Coss. & Germ., cv. CDC Robin, bought from the organic farm Floriddia (Pisa, Italy), were surface-sterilized for 3 min in 5% sodium hypochlorite and, after washing, they were germinated in glass Petri dishes (30 seeds x 10 dishes for control and each treatment) at 24 °C ± 1 °C, under light intensity of 300 μmol m⁻² s⁻¹ following a 12 h/12 h day/night cycle with a relative humidity of 70%. The different treatments comprised: distilled water (control, C), a suspension of 50 mgL^-1 TiO₂ nanoparticles (nano-TiO₂), a suspension of 50 mgL^-1 polystyrene nanoplastics (nano-PS), a suspension of 50 mgL^-1 TiO₂ nanoparticles and 50 mgL^-1 polystyrene nanoplastics (nano-TiO₂+nano-PS). Commercial powder of nano-TiO₂ was bought from US Research Nanomaterials Inc. (Houston, USA) as anatase crystal phases (nominal size of 30 nm); nano-PS were purchased from Fisher Scientific S.A.S. as polystyrene green fluorescent particles (nominal size of 30 nm). After five days the length of roots and shoots was recorded, and after measurement seedlings were collected and washed. Roots and shoots were isolated and stored at -80°C or fixed as specified below.

Suspensions of nano-TiO₂, nano-PS and of their combination, at the selected concentration, were used for TEM observations after an interaction time of 72 h from their preparation. A drop (10 μl) of each suspension was placed on TEM grids covered with formvar, allowed to settle and to dry. The grids were observed under a FEI Tecnai G2 Spirit electron microscope at 100 kV, to evaluate morphology, size, and possible aggregation of isolated and mixed particles.

Roots from control and treated seedlings were collected after three days of seed imbibition and fixed overnight in ethanol/glacial acetic acid (3: 1 v/v). Root tips were stained following Feulgen procedure and squashed on microscope slides as described in a previous paper (Giorgetti et al., 2011). For each thesis, 3 slides were analyzed, counting at least 1000 nuclei per slide. The material was examined both under a light microscope and under fluorescence at 560 nm, wavelength specific for pararosaniline (Böhm and Sprenger, 1968) with a Zeiss Axio Observer Z.1, equipped with Zeiss Axiocam MRm. The cytological analysis was done to evaluate the mitotic index, (MI, number of mitoses per 100 nuclei), the frequency of the different mitotic phases and the total cytogenetic anomalies (number of aberrations per 100 nuclei).

The determination of hydrogen peroxide content in roots and shoots was determined following the method outlined by Jana and Choudhuri (1982). Plant material was homogenized in 50 mM phosphate buffer, pH 6.5, and centrifugated at 6000g for 25 minutes. H₂O₂ content was assessed by mixing the supernatant with 0.1% titanium chloride in 20% (v/v) H₂SO₄ and measuring the absorbance at 410 nm. The H₂O₂ concentration was determined using a standard curve and expressed as μmol g⁻¹FW.

For the estimation of lipid peroxidation, TBARS (thiobarbituric acid reactive substances) were measured as in Spanò et al. (2017). The concentration of TBARS was quantified as nmol g⁻¹ FW, measuring specific absorbance at 532 nm and subtracting non-specific absorbance at 600 nm. Calculations were performed using an extinction coefficient of 155 mM⁻¹ cm⁻¹.

Total phenols were measured using Folin-Ciocalteu reagent, according to Arezki et al. (2001). Phenolic extracts obtained after homogenization of plant material in HCl 0.1 N, were added to Folin-Ciocalteu reagent, left so for 3 min in the dark, and incubated at 100°C for 1 min in the presence of Na₂CO₃ (20% w/v). After cooling, the absorbance at 750 nm was read. Level of phenolic compounds was expressed as equivalent of gallic acid (GAE mg g^-1 FW) on the base of a standard calibration curve.

For antioxidant enzymes, extraction was made as in Spanò et al. (2013) after grounding of plant material in liquid nitrogen and homogenization in 100 mM potassium phosphate buffer (pH 7.5). Ascorbate peroxidase (APX, EC 1.11.1.11) activity was measured according to Nakano and Asada (1981) following the decrease in absorbance at 290 nm (extinction coefficient 2.8 mM⁻¹ cm⁻¹) as ascorbate was oxidized. Catalase (EC 1.11.1.6) activity was determined according to Aebi (1984) and calculated using the 39.4 mM⁻¹ cm⁻¹ extinction coefficient for hydrogen peroxide. Guaiacol peroxidase (POX, EC 1.11.1.7) activity was determined as described in Arezki et al. (2001) determining guaiacol oxidation by H₂O₂ at 470 nm (extinction coefficient 26.6 mM⁻¹ cm⁻¹), one unit oxidizing 1.0 μmol guaiacol per min. The assessment of all enzymatic activities was conducted at 25°C and expressed as U mg^-1 protein. Protein quantification was carried out according to the method described by Bradford (1976), using bovine serum albumin (BSA) as a standard.

Electrophoresis was performed on 10% PAGE as in Sorce et al. (2017), using Tris-HCl 1.5 M pH 8.8. After extraction from roots and shoots, equal amounts (15 μg) of proteins were loaded onto electrophoretic gel. After running (200 V, constant current of 35 mA gel^-1), gels were incubated (in the dark, for 90 min) in 1 M Na-acetate buffer pH 4.6 containing 0.04% benzidine and 10 mM H₂O₂. POX activity was visualized as dark brown bands.

Ten roots and shoots, each of similar size and length, sourced from randomly chosen seedlings for each treatment, were isolated, and sliced using a hand microtome. The roots were dissected approximately 3–4 mm from the tip, while the shoots were cut at the beginning of the first internode.

Control samples underwent a general organ anatomical assessment by staining slices with toluidine blue (Bartoli et al., 2017).

Amplex UltraRed Reagent (Life Technologies, USA) was applied to root cross sections for in situ detection of H₂O₂ (Ruffini Castiglione et al., 2016). After staining, slices were mounted in glycerol and observed with fluorescence microscope (568ex/681em nm). BODIPY 581/591 C11 was applied as a fluorescent marker to visualize lipid peroxidation levels with a change of the fluorescence emission peak from red to green (Spanò et al., 2022). Microscope analysis was performed acquiring simultaneously the green (485ex/510em nm) and the red fluorescence (581ex/591em nm) signals and merging the two images.

To avoid interference with the red autofluorescence of chlorophyll, for the shoot we used probes whose signal was detectable by light microscopy. In situ localization of hydrogen peroxide in shoot was performed by 3,3′-diaminobenzidine (DAB) staining (Spanò et al., 2019). This compound is oxidized by hydrogen peroxide occurring in plant cell/tissues to generate dark-brown precipitates. Shoot cross sections were soaked in a freshly prepared incubation medium containing 1 mg mL⁻¹ DAB for 4 h at 25°C in complete darkness, then lightened in 96% ethanol at 65°C for 60 min. After extensive rinsing, the samples were immediately observed under light microscopy. Histochemical determination of lipid peroxidation was performed with Schiff’s reagent (Menicagli et al., 2022) (VWR Chemicals BDH) that, binding to free aldehyde groups, can be considered a qualitative indicator of lipid peroxidation. Shoot cross sections were incubated with the dye for 60 min at room temperature, then bleached in 96% ethanol for 60 min at 65°C and analyzed under light microscope to evaluate the developed purple color. Utilizing nano-PS labeled with a green fluorophore enabled us to trace their presence within various root and shoot compartments by examining cross-sections under a fluorescent microscope (468ex/508em nm).

Fluorescence microscope analyses were carried out with a Leica DMLB, equipped with appropriate sets of excitation/emission filters and with a Leica DFC7000 T camera. Optical microscope analyses were performed with a Leitz Diaplan microscope, and images were captured using a Leica DFC 420 camera.

The data were expressed as the mean ± standard error (SE), derived from a minimum of four replicates, except when otherwise specified. A significance threshold of P < 0.05 was employed. One-way analysis of variance (ANOVA) was carried out, followed by Tukey’s multiple comparison post-hoc test.

To describe the anatomy of lentil, root and shoot sections were analyzed after Toluidine Blue staining. Root cross-sections ( Figure 1A ) were performed in the maturation area, enabling the differentiation of various layers from the outermost to the innermost: rhizodermis with prominent root hairs, exodermis, cortical parenchyma, and endodermis. The central cylinder exhibited organization with a pericycle defining a triarch stele.

Shoot cross-sections ( Figure 2A ) allowed to distinguish the epidermic layer, cortical sclerenchyma, cortical parenchyma with cortical bundles, typical of species with winged or grooved stem. Vascular stele appeared in a transition structure not yet organized in a true eustele.

The utilization of nano-PS labelled with fluorophores enabled us to monitor their presence within various compartments of both roots and shoots in our model plant. In root samples treated with nanoplastics, a faint and spread-out fluorescent green signal was observed, intensifying notably in integumental and vascular tissues, especially for cotreated samples ( Figures 1J, M ). Nano-PS were translocated from the root to the shoot, the green fluorescent signal being clearly visible in sections of samples treated with nanoplastics as such or in cotreatment, the latter with greater green color intensity ( Figures 2J, M ). No fluorescent signal was detectable in sections obtained from samples treated with water (C, Figures 1 , 2D ) or with metal nanoparticles alone (nano-TiO₂, Figures 1 , 2G ).

Isolated nano-PS showed regular, almost rounded shape and diameters ranging from 20 to 50 nm. They appeared mostly dispersed, rarely forming aggregates of at most 3 particles ( Figure 3A ).

Isolated nano-TiO₂ were extremely heterogeneous in shape. They generally appeared prismatic in shape, with size ranging from 20 to 80 nm. They formed large aggregates and rarely were observed isolated ( Figure 3B ).

The suspension containing the mixture of both particles ( Figures 3C, D ) showed no evident interaction between the two types of nanoparticles. In particular, the nano-PS appeared randomly dispersed and not adjacent to the aggregates of the nano-TiO₂, both keeping the same characteristics as when observed isolated ( Figures 3C, D ).

The effects of treatments on growth were different in the different organs. Only nano-PS, also in cotreatment with nano-TiO₂, induced a significant inhibition in root length ( Table 1 ), while in shoots ( Table 1 ) both nano-TiO₂ and nano-PS, also in cotreatment, significantly reduced growth. Interestingly, the highest reduction in length was always recorded in seedlings treated with nano-PS.

Root meristems of the primary lentil root were analyzed to determine the possible cytogenetic abnormalities induced by the different treatments taking into account mitotic index and the disturbance of mitotic phases in terms of c-metaphases, abnormal anaphases with chromosomal bridges and fragments and abnormal mitotic spindles.

The analysis of the mitotic index (MI) showed a decrease of the frequency of cell divisions in treatments with nanoparticles compared to the control (C, MI = 6.73%; nano-TiO₂, MI = 5.86%; nano-PS, MI = 4.97%, nano-TiO₂+ nano-PS MI = 2.26%) ( Figure 4A ). The differences were greater and statistically significant in the treatment with the two types of nanoparticles in combination, nano-TiO₂ + nano-PS, where the reduction was approximately 66.4% compared to the control ( Figure 4A ).

The analysis of mitotic phases frequencies is shown in Table 2 . No significant effects were found concerning the frequency of prophase, but abnormal metaphases and anaphases significantly increased in treated root meristems with the maximum reached for nano-TiO₂ + nano-PS (abnormal metaphases 28.29%, abnormal anaphases 20.46%). The increase in the total cytogenetic abnormalities was clearly shown in Figure 4B . In particular, the cotreatment with nano-TiO₂ and nano-PS induced the greatest number of anomalies (49.45%), much higher than the treatment with nano-TiO₂ (32.8%) and nano-PS (42.96%) alone. Some representative examples of cytological anomalies are shown in Figure 5 . Unlike the control, in which mainly normal mitoses were observed ( Figures 5A-E ), in all treatments with nanoparticles it was possible to observe sticky chromosomes ( Figures 5F, K, L ), c-metaphases (colchicine-like metaphases, caused by partial or complete spindle failure at mitosis) ( Figures 5G, P ), abnormal anaphases with bridges ( Figures 5H-J ), lagging chromosomes ( Figures 5M-O ) and abnormal mitotic spindles ( Figures 5I-J, Q, R ). Moreover, in nano-TiO₂ + nano-PS treatment, other cytological abnormalities were observed in the cytoplasm of both mitotic and interphase cells, such as the presence of Feulgen-positive bodies ( Figures 5S, T, U ), the presence of micronuclei ( Figure 5V ) and cytoplasmic vesicles ( Figures 5W, X, Y ).

In our experimental conditions, the concentration of hydrogen peroxide ( Figure 6A ) was significantly higher in shoots and roots of seedlings treated with nano-PS, both alone and in cotreatment with nano-TiO₂. The highest values were always detected under nano-PS treatment. Oxidative damage, estimated as TBARS ( Figure 6B ), was generally lower in treated seedlings in comparison with controls. Only in roots cotreatment induced the highest damage, significantly higher than that recorded in all other treatments. Histochemical probes, proposed specifically for hydrogen peroxide and lipid peroxidation, as illustrated in Figures 1 and 2 , allowed to identify qualitative signals indicative of oxidative stress. In the cross sections of both control and treated roots, the red fluorescent probe Amplex, specific for H₂O₂, yielded a signal mostly localized within xylem vessels, with varying intensity staining observed in the root epidermis ( Figure 1 ). In nano TiO₂-treated samples, the red signal extended to include phloem arches ( Figure 1E ), while nanoplastic-treated samples exhibited staining, particularly noticeable in the tegumental tissues and partially covering the cortex ( Figure 1H ). Cotreatment resulted in a heightened response within the vascular cylinder, extending nearly across the entire cortical cylinder, being the most responsive treatment to Amplex staining ( Figure 1K ). Cotreated sample exhibited the highest reactivity also in relation to the BODIPY fluorescent probe, as depicted in Figure 1L . This probe identifies lipid peroxidation by detecting a shift in fluorescence emission from red to green. In the cotreated sample, green fluorescence was consistently observed in all root tissues, encompassing both cortical and vascular cylinder regions ( Figure 1L ). In the other samples, the signal was weakly diffused over the whole section ( Figures 1F, I ), with the control showing specifically a general signal absence in the vascular tissues ( Figure 1C ).

The histochemical assessment of hydrogen peroxide (H₂O₂) using DAB in shoot cross sections of control samples indicated positive signals, as brown precipitates, concentrated in the xylem of both cortical bundles and stellar vascular bundles ( Figure 2B ). Treated samples exhibited a similar staining pattern, albeit more diffuse within the shoot ( Figures 2E, H, K ), with the enhancement being particularly notable in plants treated with nano-PS ( Figure 2H ).

The in situ detection of lipid peroxidation using Schiff’s reagent, as a magenta color development, did not reveal distinct differences in the staining pattern ( Figures 2F, I, L ), except for the tegmental tissues that demonstrated heightened responsiveness in samples treated with nano-TiO₂, either alone or in cotreatment with nano-PS ( Figures 2F, L ).

The highest concentration of phenols was measured in seedlings treated with nano-PS, though only in shoot the value was significantly higher than in controls ( Tables 3 , 4 ). In root, APX and CAT activities were lower in all treatments than in control ( Table 3 ). Nano-PS-treated plants were characterized by the lowest catalase activity. An opposite trend was recorded for POX activity, that was higher in treated plants in comparison to control, though the differences were significant under nano-PS alone or in cotreatment with nano-TiO₂. In shoot there were no differences among control and different treatments, with the only exception of CAT activity, that was significantly higher in nano-TiO₂-treated plants than in all other materials ( Table 4 ). After electrophoresis ( Figure 7 ), specific staining for revealing POX activity highlighted bands with different mobility, with distinct patterns depending on the organ, but not on the different treatment. Noteworthy is the presence of a band with greater mobility detectable only in the roots, in accordance with the higher activities recorded in this organ in comparison with shoots.