Evaluation of morpho-physiological responses and genotoxicity in Eruca sativa (Mill.) grown in hydroponics from seeds exposed to X-rays

Seed irradiation and plant culture condition

Seeds of Eruca sativa were irradiated by 1 and 10 Gy X-rays at the Department of Physics (University of Napoli Federico II, Napoli, Italy) by means of a radiogenic tube (STABILIPAN II; Siemens, Berlin, Germany) at the Radiation Biophysics Laboratory. Doses were administered through a 1-mm Cu filtration at a dose rate of about 1.36 Gy min⁻¹. Dosimetry checks were routinely performed by an ionization chamber to ensure dose uniformity within a square field of 15 cm side. Irradiated seeds and non-irradiated seeds used as control were germinated on wet filter paper in the dark at 25 °C for 4 days. When the roots emerged and the cotyledons appeared fully developed, 15 seedlings for each treatment and control were transplanted to a hydroponic system (Sorrentino et al., 2021) containing 5 L of nutrient solution (Hoagland & Arnon, 1950) at pH 5.8 (Fig. S1). The growth chamber was settled at the following conditions: (1) temperature 24/18 °C; (2) relative humidity (RH) 55–75% (day/night); (3) photoperiod of 16 h light per day with a photosynthetic photon flux density (PPFD) at the top of the canopy of 180–190 μmol photons m⁻² s⁻¹. All plants were analyzed after 30 days culture.

Morphological traits, stomata counting and sizing

Length and weight of the root and shoot, and leaf number were considered in 10 plants after 30 days culture for each treatment and control for morphometrical analysis. Stomata number was determined on five fully expanded leaves for each treatment. Two surface replicas were obtained by nail topcoat at midlamina of the leaves. For each treatment 10 surface areas of about 160,000 µm² were observed under a light microscope (for a total analyzed surface of 1.6 mm² per treatment) and images acquired were analyzed by ImageJ software (National Institute of Health, Bethesda, MD, USA).

Photosynthetic pigment content

Chlorophyll a, chlorophyll b and carotenoid contents were determined in five leaves (from different plants) per treatment following the procedure described by Lichtenthaler (1987). In brief, pigments were extracted from fresh leaf tissue previously weighed (200–300 mg) by mortar and pestle in ice-cold 100% acetone and centrifuged at 5,000 rpm for 5 min (Labofuge GL; Heraeus Sepatech, Hanau, Germany). The absorbance of supernatants was quantified by spectrophotometer (Cary 100 UV-VIS; Agilent Technologies, Santa Clara, CA, USA) at 470, 645 and 662 nm and pigment concentrations expressed in µg g⁻¹ FW, calculated as follows:

• [Chl a] = 11.24 * A₆₆₂ – 2.04 * A₆₄₅

• [Chl b] = 20.13 * A_{645 −} 4.19 * A₆₆₂

• [Chl a + Chl b] = 7.05 * A₆₆₂ + 18.09A₆₄₅

• [c + x] = (1000 * A₄₇₀ − 1.9 * [Chl a] − 63.14 * [Chl b])/214

where [Chl a], [Chl b] and [c + x] are the concentrations of chlorophyll a, chlorophyll b, and total carotenoids, respectively.

Protein analysis

Photosynthetic protein analysis was carried out because protein pattern may be severely affected by abiotic stress, including radiation (e.g., Arena et al., 2013). Protein extraction of leaves was carried out on plants after 1-month growth using 0.3 g of plant material for each sample (Bertolde et al., 2014; Wang et al., 2006). An SDS-PAGE (10%) was performed by using Dual Color Protein Standard (Bio-Rad, Hercules, CA, USA) as marker and Laemmli loading buffer added to the samples to follow protein separation. Western blot analysis on protein samples was performed using a blocking solution (100 mM Tris-HCl pH 8.0, 150 mM NaCl, 0.1% Tween 20, 5% BSA) and primary specific antibodies (Agrisera) to reveal the Rubisco (anti-RbcL, rabbit polyclonal serum), and the Actin (anti-ACT, rabbit polyclonal serum), used as loading control. The immunorevelation was performed using the kit for chemiluminescence (Westar Supernova; Cyanagen, Bologna, Italy) by ChemiDoc System (Bio-Rad, Hercules, CA, USA). Densitometry analysis was performed following Sorrentino et al. (2018) using ImageJ software (Rasband, W.S., U.S. NIH, Bethesda, MD, USA, 1997–2012), normalizing each Rubisco band value to the corresponding actin band value. Results were expressed as percentages of the control set to 100%.

Antioxidants

The antioxidant analysis was carried out by the ferric reducing antioxidant power assay (FRAP). Powdered fresh leaf samples 250 mg each (homogenized in a TissueLyser LT after freezing in liquid nitrogen), were mixed with 60:40 (v/v) methanol/water solution and centrifuged at 14,000 rpm for 15 min at 4 °C. Then, acetate buffer was added to the extract (1:16 300 mM pH 3.6); The acetate buffer contained a mix of tripyridyltriazine (TPTZ) (10 mM TPTZ in 40 mM HCl, 1:1.6) and FeCl₃ (1:16 12 mM FeCl₃). After 1 h of incubation at 4 °C, the absorbance was measured at 593 nm with a spectrophotometer (UV-VIS Cary 100; Agilent Technologies, Palo Alto, CA, USA) using Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid) as standard. The antioxidant capacity was expressed as μmol Trolox equivalents for mg of fresh sample.

Phenols

The assay by Folin–Ciocalteu (F–C) reagent was used to quantify the total phenolic compounds (Singleton & Rossi, 1965). The F–C assay is based on the oxidation of phenols in alkaline environment, with the transfer of electrons to phosphomolybdic/phosphotungstic acid complexes, whose reduced form appears blue. Phenolic concentration was determined by a spectroscopy at 760 nm. Although the electron transfer reaction is not specific for phenolic compounds, the extraction procedure eliminates approximately 85% of ascorbic acid and other potentially interfering compounds (Ainsworth & Gillespie, 2007).

In brief, 1 ml of 60% methanol was added to 250 mg of ground fresh tissue (homogenized in a TissueLyser LT after freezing in liquid nitrogen) in a 1.5 ml tube. The samples were mixed by vortexing and placed on ice for 3 min in the dark and then transferred into a 15 ml tube; the volume was brought to 5 ml with the addition of 60% methanol. The samples were then centrifuged at 3,000g for 5 min. Then 62.5 μl of supernatant were added to 62.5 μl of Folin-Ciocalteu’s reagent (Sigma, St. Louis, MI, USA) and 250 μl of deionized water and incubated for 6 min at room temperature. After the addition of 625 μl of sodium carbonate 7.5% and 500 μl of deionized water, the samples were incubated for 90 min at room temperature. The absorbance was measured at 760 nm.

Total phenolic was expressed in μg of gallic acid equivalent/mg of fresh weight, based on the standard curve of gallic acid drawn on a dynamic range between 0 and 125 μg of gallic acid.

Genotoxicity

Three young leaves (15 d), at the same development stage, were selected from three plants for each treatment (control, 1 and 10 Gy X-ray irradiation), and DNA-extracted and amplified following Sorrentino et al. (2022). In brief, total genomic DNA was extracted by using a modified CTAB (cetyl-trimethyl ammonium bromide) method (Murray & Thompson, 1980). DNA amplification was performed by using six different inter simple sequence repeat (ISSR) primers (Table S1).

Polymerase chain reactions (PCRs) were carried out three times for each primer to confirm the reproducibility of banding profiles; however, only constant bands were selected for the analysis and Genome template stability (GTS) calculation. Each amplification reaction contained DNA (15 ng), ddH₂O, 10X Dream Taq Buffer, 1 mM MgCl₂, 0.2 mM each dNTP, 0.6 mM primer, and 1 U Dream Taq DNA polymerase in a final volume of 25 µL (Thermo Fisher Scientific, Waltham, MA, USA).

The thermocycler (Esco Swift Maxi Thermal Cycler) was programmed for an initial denaturation step at 94 °C for 3 min, followed by 35 cycles with 1 min at 94 °C, 1 min annealing and 2 min extension, and a final extension cycle of 7 min at 72 °C. Amplification were run by electrophoresis on 2% agarose gel with Tris-borate-EDTA (TBE) buffer; amplification products stained with GelRed Nucleic Acid 10,000 (biotium) were visualized under UV light using GelDoc (Bio-Rad, Hercules, CA, USA). Size estimates of the ISSR bands were made using a 100-bp ladder. ISSR profiles (i.e., bands between 200 and 1,300 bp) for each primer were analyzed by using the software GelAnalyzer 2019 for band counting and intensity assignment. Changes in the ISSR patterns were expressed as a decrease in GTS, a quantitative measure of DNA stability based on ISSR banding of treated samples compared to the control samples. The GTS was calculated by the formula:

(1) $$\mathrm{G}\mathrm{T}\mathrm{S}=[1-\mathrm{a}/\mathrm{n}]\times 100$$

where a is the average number of polymorphic bands in each treated sample and n the number of all bands in the control. Therefore, GTS values expresses the percentage of non-polymorphic bands. Firstly, genetic variation was also estimated among the three control plants to establish within-control variation as a threshold to evaluate the eventual occurrence of the genotoxic damage. Then, DNA profiles of one control plant was arbitrarily fixed as a reference control plant to be compared to DNA profiles of the plants from X-ray-treated seeds. The differences in band intensity were also calculated by GelAnalyzer software in comparison to the bands of the 100 bp ladder (here used as gel marker), having known DNA concentrations; only difference of intensities higher than 20% were considered. While comparing treated to control profiles, only changes observed in all the three replicates of the treated plants were considered. The GTS was calculated for each treatment and expressed as a percentage of the non-variable bands compared to the total band number. The entire procedure above described was repeated for mature leaves (three 30 day leaves for each treatment) to check a possible recovery of the genotoxic damage.

Data analysis

Normality and homogeneity of the variance of the data were assessed by the Shapiro–Wilk and Levene test, respectively. Data were analyzed with one-way ANOVA and, for count data, with Generalized Linear Model with a Poisson distribution, using IBM SPSS Statistics for Windows (IBM Corp. Released 2020, Version 27.0; IBM Corp, Armonk, NY, USA). Multiple comparison tests were performed with Tukey’s post-hoc test (p < 0.05).

Analysis of morphological traits

The germination percentage was always 100% for the control, 1 and 10 Gy treatments. Morphological traits were differently affected by irradiation (Table 1); some traits were more susceptible than others to the treatments. Specifically, plant height did not differ between control and treated plants. Conversely, the number of the leaves and weight of the shoots increased at 1 Gy compared to control, although this increment was significant only for the shoots; the average shoot weight measured in these plants was indeed the highest (i.e., 12.8 g). Roots were significantly longer in plants grown from 10 Gy irradiated seeds, with an average length of 23.1 cm. Root weight was instead significantly lower in plants developed from irradiated seeds, with a decrease of about 33% observed in 1 Gy treated plants and even higher under 10 Gy treatment.

Stomata counting and surface

Stomata number (Fig. 1) significantly increased under IR stress compared to the control plants from an average of 177 for control plants to 313 in plants treated with 10 Gy, at parity of surface analyzed (160,000 µm²). A significant increase in stomal surface compared to control was also observed in plants treated with 1 and 10 Gy X-rays; particularly, stomal surface about doubled in 1 Gy treated plants, whereas it increased of 30% in 10 Gy treated plants. Hence,, this increment, also evident by observing the images under the light microscope (Fig. 2), was not dose-dependent but followed a hormesis trend (Fig. 1).

Pigment content

Although the absence of chlorosis and necrosis in the leaves of the plants from irradiated seeds, a dose dependent decrease was found for Chl contents, statistically significant at the highest dose of 10 Gy (Fig. 3). In fact, in these leaves Chl a decreased more than 30% compared to control (from 1.03 to 0.65 µg g⁻¹ F.W.). Similarly, a significant decrease of Chl b was observed under both treatments, especially in leaves from 10 Gy treated samples. Total chlorophyll content (Chl a+b) enforced this trend, showing a significant decrease in both treatments compared to control. The Chl a/Chal b ratio (Fig. 3) was not significantly different among samples, probably due to the high variability observed in 10 Gy leaves. The sum of xanthophyll and carotenoids (x+c) showed a dose dependent statistically significant decrease from control to 10 Gy dose, as well.

Protein analysis

Protein analysis of the leaves showed a reduction of the Rubisco concentration with both tested doses, compared to control plants, whose content was arbitrary set equal to 1. However, the decrease was more pronounced at 1 Gy, with a concentration of Rubisco about 24% of the control, while at 10 Gy a concentration of Rubisco of about 76% compared to the control plants was measured (Fig. 4).

Phenols and total antioxidant content

The 1 Gy dose determined a significant increase in phenol content of about 30% compared to control plants (from 57 to 86 µg) gallic acid equivalent per mg tissue (F.W.), while at 10 Gy dose phenol content resulted comparable to control (Fig. 5). Although an increasing trend was observed with the increasing dose no statistically significant differences were found among the total antioxidant contents of control plants compared to plants grown from seeds exposed to ionizing radiations; total antioxidant contents ranged from 0.93 (control) to 1.12 (10 Gy) µmol Trolox equivalent mg⁻¹ F.W. (Fig. 5).

Genotoxicity

A total of 48 reproducible bands were found in the control 15 d and 30 d leaves. The genome template stability (GTS) percentage (Table 2; Fig. S2) calculated in the control plants was 91%, which means that the 9% of the examined bands showed variations, mostly in form of increased/decreased band intensity (i.e., variation of type c and d). X-rays induced a noticeable decrease of the GTS percentage that halved under 1 Gy treatment and even more under 10 Gy, in the 15 d-leaves from irradiated seeds. In 1 Gy treated samples the higher contribution to the reduction of the GTS was given by the appearance and disappearance of bands (about 30%); whereas, similarly to control, in the 10 Gy treated samples most decrease of the GTS depended on the variation in band intensity (about 35%). In 30 d-leaves a partial recovery of the GTS percentages was observed in plants from irradiated seeds, more pronounced in 1 Gy plants, with a GTS reaching 69% and 56% in 1 and 10 Gy plants, respectively (Table 2).