Location of the experiment and plant material

The research was carried out at the Center for Protected Agriculture of the Faculty of Agronomy of the Autonomous University of Nuevo León, Mexico, inside a Spanish-type greenhouse. The average environmental conditions inside the greenhouse were 25 °C temperature and 70% relative humidity. Eight-month-old fig plants, with six leaves each, were transplanted into black polyethylene bags of a capacity of 10 L filled with perlite substrate. The experiment was established under a completely randomized experimental design with a 3x2x2 factorial arrangement: three fig cultivars (Ficus carica L.): Brown Turkey (BT), Kadota (K) and Black Mission (BM); two levels of NaCl (0 and 100 mmol L^-1) and two levels of I (0 and 10 mg L^-1) using potassium iodate (KI) as a source of iodine. Both factors, NaCl and I were applied by gravity irrigation.

The base irrigation water had the following characteristics: Ca: 3.5 Mg, 1.8, Na: 0.9, HCO₃: 2.5, Cl: 1.35, SO₄: 2.35; HCO₃: 2.5 mEq L^-1; pH: 7.5 and electrical conductivity (EC): 0.6 dS m^-1. From the combination of the aforementioned factors, 12 treatments emerged, with six repetitions each. Each plant was considered as an experimental unit. The relative chlorophyll content, expressed in SPAD units, was measured using the portable SPAD-502 of Minolta (Konica Minolta, Osaka, Japan). SPAD units were measured in the middle lobe of leaves number 4, 5 and 6 from the base to the apex.

The values of this variable are the average of three measurements made 5, 10 and 15 days after the beginning of treatments. Also, after the last measurement, samples of four plants per treatment were collected for the determination of dry weight of new leaves, antioxidant compounds and pigments. The dry weight was obtained by means of a drying oven (Yamato model DX602C, Yamato Scientific America, Santa Clara, CA, USA) where the samples were subjected to a temperature of 60 °C for three days. The resulting material was weighed with an analytical balance with precision of 0.001 g.

Extraction of phenolic compounds

The extraction of phenolic compounds was carried out based on what was reported by ^{Carballo-Méndez et al. (2019}) with slight modifications. Four grams of fresh leaf tissue were suspended in 40 mL of water:methanol (20:80 v/v) (80%) and homogenized with an Oster blender (M4655-813/465-42, Sunbeam, Mexico City, Mexico) in a crystal glass for porridge for 30 s. It was then filtered with an organza fabric, placed in 50 ml Corning tubes and centrifuged at 4 500 rpm for 5 min at 25 °C. The supernatant was recovered and stored protected from light at -20 °C, until its further analysis.

Total phenols and flavonoids

The content of total phenols and total flavonoids was evaluated based on what was reported by ^{Rodríguez-Salinas et al. (2020}), with the following modifications: the total phenols were evaluated based on the colorimetric method of the Follin-Ciocalteu colorimetric reaction, with 200 μl of the methanolic extract in 2 600 μl of water, followed by the addition of 200 μl and alkalinization by adding 2 000 μl of Na₂CO₃, then it was left to react for 90 min and the absorbance was measured in an SP-830 Plus spectrophotometer (Barnstead, Turner, USA) at a wavelength of 750 nm.

The standard curve was prepared with gallic acid in concentrations from 0 to 200 mg L^-1, 80% methanol was used as a blank. The results were expressed in milligrams of gallic acid equivalents per gram of fresh tissue (mgGAE g^-1). The total flavonoids were measured by the method of aluminum chloride (AlCl₃) and sodium hydroxide (NaOH), briefly, 200 μl of the methanolic extract was added to 3 500 μl of water, followed by 150 μl of a 5% solution of NaNO₂ and it was left to stand for 5 min, then 150 μl of a 10% solution of AlCl₃ was added and finally 5 min later, 1 000 μl of NaOH at 1 N was added.

It was left to react for 15 min and the absorbance of the samples was measured in an SP-830 Plus spectrophotometer at the length of 510 nm. Concentrations were calculated based on a catechin curve made at concentrations from 0 to 200 mg L^-1. The results were expressed in milligrams of catechin equivalents per gram of fresh tissue (mg CE g^-1).

DPPH antioxidant capacity

The determination of antioxidant capacity by the method of 2,2-diphenyl-1-picrylhydrazyl (DPPH) of the methanolic extracts of the fresh fig leaf tissue was carried out based on what was described by ^{Rodríguez-Salinas et al. (2020}), with slight modifications described below: DPPH, it was evaluated using the working stock solution prepared at 60 μM dissolved in 80% methanol, adjusted to an absorbance at 1 nm at a wavelength of 517 nm. The reaction was performed by adding 0.5 μl of the methanolic extract to 1.5 ml of the stock solution, it was left to stand for 30 min and the absorbance was measured on an SP-830 Plus spectrophotometer at a wavelength of 517 nm. As a standard curve, Trolox was used in concentrations from 0 to 500 μmol L^-1. Results were expressed in Trolox equivalents per each gram of fresh tissue (μmol TE g^-1).

The IC50 of DPPH was calculated for the amount of antioxidant needed to inhibit 50% of the oxidation of the radical, the absorbance adjusted to 1 in the working solutions was considered as 100% of oxidation and methanol was used as a control. The results were expressed in milligrams per gram of fresh leaf tissue (mg g^-1).

Photosynthetic pigments

The photosynthetic pigments chlorophylls (a, b and total) and total carotenoids were extracted and calculated based on what was reported by ^{Rajput and Patil (2017}), with slight modifications. Briefly, on average 4 g of fresh leaf tissue was homogenized in an Oster blender with a crystal glass for porridge for 30 sec. in the presence of 40 ml of water:acetone (20:80 v/v) (80%) that contained 200 mg of Na₂CO₃. The homogenized was then passed through an organza fabric, placed in 50 ml capacity corning tubes and centrifuged at 6 000 rpm for 5 min at room temperature. The supernatant was recovered and stored protected from light at -20 °C, until its subsequent analysis by spectrophotometry.

Chlorophylls and total carotenoids were quantified in an SP-830 Plus spectrophotometer at wavelengths of 663-645 and 480-510 nm, respectively. The results were expressed in milligrams per gram of fresh tissue (mg g^-1).

;

;

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Where: A= absorbance obtained at the specified wavelength; V= volume of extraction; and SW= sample weight.

Statistical analysis

The values obtained were analyzed with the Levene test for homogeneity of variances and with the Kolmogorov-Smirnov test for the verification of normality. For the analysis of variance of the variables, the relative chlorophyll content covariate was considered, which was evaluated one day before the start of the treatments. In the variables where a significant difference was found, a comparison of means was made using the Tukey test (p< 0.05). For these analyses, the statistical package SPSS version 22.0, IBM, was used.

Phenolic compounds and antioxidant capacity

The concentration of phenolic compounds was affected by the interaction of the different factors. The BR and K cultivars reduced the concentration of total phenols by 26 and 45% in the absence of NaCl, while with the presence of NaCl, these compounds increased by 25 and 17%, respectively. Contrary to the above, in the BM cultivar, the presence of I in saline situation reduced total phenols by 9% (Figure 2A). The concentration of total flavonoids was only affected by cultivars, where the BT cultivar exceeded K and BM; in general, 150% (Figure 2B). The antioxidant activity by DPPH showed interaction between the three factors. In the BT cultivar, the presence of I in the solution without NaCl decreased this activity by 11%, while in the presence of NaCl, I increased it by 455%. In the BM cultivar without the presence of NaCl, the presence of I increased this activity by 22% (Figure 2C).

The IC50 was only affected by the interaction of cultivars and I, particularly in the BM cultivar where the presence of this element reduced this concentration by 21% (Figure 2D). The increase in antioxidant capacity by the DPPH assay of plants reveals an increase in the synthesis of low molecular weight reducing molecules, mainly non-enzymatic antioxidants (^{Pérez-Salas and Medrano-Macías, 2021}), such as phenolics (^{Leyva et al., 2011}). Because DPPH bases its foundation on the ability to measure compounds with the ability to donate H atoms (^{Schaich et al., 2015}).

Figure 2 Effect of cultivars, NaCl and I on the concentration of total phenols (A); total flavonoids (B); DPPH antioxidant activity (C); and mean inhibitory concentration (IC50). The lines on the bars represent the standard error. The different letters on each bar mean that the treatments were statistically different (Tukey, p< 0.05). 

Several authors have reported that applications of I increase the antioxidant capacity of plant tissues. ^{Kiferle et al. (2019}) mention that I increased the total phenols in basil leaves, as well as ^{Blasco et al. (2013}), who reported that, in lettuce subjected to salt stress, I increased the concentration of total phenols, without finding differences in the concentration of flavonoids. These results are consistent with what was observed in this experiment. Contrary to the above, ^{Golubkina et al. (2018}) reported that, in mustard (Brassica juncea L.) leaves, the application of I increased the concentration of flavonoids.

Similarly, ^{Cortés-Flores et al. (2016}) reported that foliar application of KI increased DPPH antioxidant activity in pepper seedlings under optimal development conditions, while ^{Kiferle et al. (2019}) reported the decrease in this activity in basil plants. In the present experiment, this effect was different between cultivars and salinity level. In non-saline conditions, the presence of iodine decreased the DPPH antioxidant activity in the BT cultivar, while it increased it in the BM cultivar. In saline situation, the presence of I only benefited the BT cultivar. Particularly for IC50, Incrocci et al. (2020) reported that, in basil, the presence of I reduced the IC50, although the impact was different between cultivars, a situation very similar to that shown in this experiment.

Photosynthetic pigments

The concentration of chlorophyll a showed main effects of the three factors. The BT and K cultivars exceeded BM by 35%. The presence of NaCl reduced these compounds by 18%, while the presence of I increased them by 12% (Figure 3A). The concentration of chlorophyll b was affected by the interaction of the three factors. In the BT cultivar, the presence of I increased them, the concentration of chlorophyll b by 17% without the presence of NaCl and by 83% in the presence of said salt. In the K cultivar, the effect of I was different since, in the absence of NaCl, chlorophyll b increased 36%, while, in the presence of salt, these compounds decreased by 17%.

For the BM cultivar, the presence of I in salinity condition increased these pigments by 65% (Figure 3B). The carotenoid concentration was only affected by the interaction between the cultivar and NaCl. In BT and K cultivars, the presence of NaCl reduced carotenoid concentration by 36% and 20%, respectively (Figure 3C). The results shown above differ partially from those reported by ^{Pérez-Salas and Medrano-Macías (2021}), who point out that, in tomato (Solanum lycopersicum L.) plants subjected to salt stress, the presence of I did not modify the concentration of chlorophyll a, but it did increase the concentration of chlorophyll b.

Figure 3 Effect of cultivars, NaCl and I on the concentration of chlorophyll a (A); chlorophyll b (B); and total carotenoids (C). The lines on the bars represent the standard error. The different letters on each bar mean that the treatments were statistically different (Tukey, p< 0.05).