Plant material and growth conditions

The experiment was conducted in a greenhouse at Antonio Narro Agrarian Autonomous University, Saltillo,Coahuila, Mexico, located at 25° 21’ 12.8’’ NL and 101° 01° 51.9’’ WL. In 2013, tomato (Solanum lycopersicum L.) var. Rio Grande seeds were sowed in 355-mL polystyrene containers. Peat moss mixed with perlite at a 5:1 ratio was used as substrate. The average greenhouse temperature was 20.7 °C, with 741 W∙m^-2 maximum radiation and 62.8 % relative humidity. Manual irrigation was carried out daily, applying 50 mL of 20 % ^{Steiner solution (1961)}. The EC and pH of the nutrient solution were 1.0 dS∙m^-1 and 6.5, respectively. General management and production practices were carried out according to the indications established by ^{Villasanti (2013)}.

The treatments consisted in the direct application of Iodine (I^-) to the substrate (S) or by foliar application (F) at a concentration of 1 μM daily (D) or 100 μM biweekly (15D). The same design was used for the potassium iodate (IO₃^-) treatments. The application began four weeks after sowing (WAS), when the seedlings had developed four leaves, and continued until sampling (eight WAS).

Only nutrient solution was applied to the substrate containing the control seedlings.

Sampling

At eight WAS, sampling was conducted under a completely randomized experimental design. Three complete seedlings per treatment were taken to determine biomass and another three for leaf quantification of enzymatic and non-enzymatic antioxidants. The experimental unit was one seedling. Analysis of variance (P ≤ 0.05), using the statistical software ^{InfoStat (2008)}, and Tukey’s range test (P ≤ 0.05) were performed with the obtained data.

Seedling analysis

Seedlings intended for biomass quantification were dried in an oven, while the leaves for non-enzymatic and enzymatic antioxidant quantification were lyophilized.

Once the plant material was prepared, the following variables were obtained:

Biomass. Seedlings were partitioned in stem, root, and leaves, and weighed using an OHAUS® digital scale. Subsequently, they were placed in a drying oven at 60 °C until constant weight. The initial and final weight was expressed in grams.

The extraction of biomolecules was made through the lyophilized seedling leaves grounded using a mortar and pestle. First, 200 mg of ground tissue, 20 mg of polyvinyl pyrrolidone and 1.5 mL of 0.1 M phosphate buffer (pH 7-7.2) were placed in a centrifuge tube, and then subjected to sonification for 5 min. Subsequently, the homogenate was centrifuged at 12,500 rpm for 10 min at 4 °C. The supernatant was collected, filtered with a nylon membrane (^{Ramos et al., 2010}) and diluted at a 1:15 ratio with phosphate buffer.

Superoxide dismutase (SOD). The activity of this enzyme from the biomolecules extract was quantified in a microplate reader using the SOD determination kit (^{SIGMA-ALDRICH, 2014}). This method is based on quantifying by spectrophotometry; the conversion of WST (water soluble tetrazolium salt) to WST-formazan dye takes place upon oxidation by superoxide ions formed by the set of xanthine (XO)/xanthine oxidase (X). The inhibition in the oxidation of WST is attributed to the neutralization of superoxide radicals caused by SOD. The units were expressed as inhibition percentage.

Catalase (CAT). Catalase activity was quantified using spectrophotometry, which was measured in two reaction times: time 0 (T0) and time 1 (T1). The control reaction mixture was prepared with 0.1 mL biomolecules extract, 1 mL phosphate buffer (pH 7.2) and 0.4 mL 5 % H₂SO₄, while the T0 reaction mixture was prepared with 0.1 mL biomolecules extract and 1 mL 100 mM H₂O₂, after which 0.5 mL 5 % H₂SO₄ were immediately added. T1 was prepared in the same manner as T0, with the exception that 0.5 mL 5% H₂SO₄ were added after 1 min of reaction between the extract and the peroxide. The reaction was conducted under constant shaking at 20 °C. Finally, H₂O₂ consumption was read at 270 nm in the UV-VIS spectrum. The activity units (UI) were expressed in H₂O₂ mM per minute by total proteins (^{Cansev, Gulen, & Eris, 2011}).

Ascorbate peroxidase (APX). First, 0.1 mL biomolecules extract, 0.5 mL ascorbate at a 10 mg∙L^-1 concentration and 1 mL 100 mM H₂O₂ were added to a centrifuge tube at 22 °C (^{Nakano & Asada, 1987}). After 1 min the reaction was terminated with 0.4 mL 5 % H₂O₂. The ascorbate consumption rate was quantified by spectrophotometry at 266 nm. The UI were expressed in mM ascorbate per minute by total proteins.

Glutathione peroxidase (GPX). This was measured using the method established by ^{Xue, Hartikainen, and Piironen (2001)}, where H₂O₂ is used as substrate. First, 0.2 mL biomolecules extract, 0.4 mL of glutathione reduced to 0.1 M and 0.2 mL 0.067 M Na₂HPO₄ were placed in a test tube. This mixture was pre-heated in a water bath at 25 °C for 5 min, then 0.2 mL H₂O₂ (1.3 mM) were added to initiate the catalytic reaction. The reaction was conducted for 10 min and stopped by adding 1 mL of trichloro acetic acid (1 %); the mixture was placed in an ice bath for 30 min and then centrifuged for 10 min at 3,000 rpm. Then 0.48 mL were taken from the supernatant and placed in a test tube, to which 2.2 mL Na2HPO4 (0.32 M) and 0.32 mL of 1.0 mmol solution of 5,5-DiThiobis(2-NitroBenzoic acid) (DTNB) were added for color development. Absorbance was measured at a wavelength of 412 nm with a UV-VIS spectrophotometer. The UI were expressed in glutathione (mM) per minute by total proteins.

Glutathione (GSH). Glutathione was quantified following the spectrophotometric technique established by ^{Xue et al. (2001)}, by the reaction with 5,5-DiThiobis(2- NitroBenzoic acid) (DTNB). First, 0.48 mL extract, 2.2 mL sodium phosphate dibasic (Na₂HPO₄ at 0.32 M) and 0.32 mL 1 mM DNTB dye were placed in a centrifuge tube. It was mixed and read in a UV-VIS spectrophotometer at 412 nm. The values were expressed in mg∙L^-1.

Ascorbate (Asa). For ascorbate extraction, 200 mg lyophilized leaves were weighed and 1 mL of solution water:acetone 1:1 was added (^{Yu & Dahlgren, 2000}). The mixture was vortexed for 30 s, then sonicated for 5 min and finally ultracentrifuged at 4 °C at 12,500 rpm for 10 min; the supernatant was removed in this process. Quantification was carried out using a Thermo Spectra system P4000® high-performance liquid chromatography (HPLC) instrument under the following conditions: 230 nm wavelength, NaH₂PO₄ mobile phase at 50 mM (pH 2.8), flow rate 1 mL·min^-1 and an AQUASIL C-18 HPLC column was used at 60 °C. The results were expressed in mg∙L^-1.

Total Phenols (Ft). Quantification was performed by UV-VIS spectrophotometry using extraction solution water:acetone 1:1 with Folin-Ciocalteu reagent. The concentration units were expressed in mg∙L^-1 (^{Nsor- Atindana, Zhong, Mothibe, Bangoura, & Lagnika, 2012}; ^{Sultana, Anwar, & Ashraf, 2009}).

Biomass

In the present study, the daily treatments received a total of 30 μM iodine and the biweekly ones 300 μM. None of the treatments caused a reduction of biomass compared to the control. Otherwise, daily applications showed increased biomass (Table 1). Reduced plant biomass is one of the clearest indicators of stress damage (^{Blasco et al., 2008}; ^{Sairam, Rao, & Srivastava, 2002}).

^{Weng et al. (2008b)} observed increased biomass in spinach grown hydroponically with I^- and IO₃^- concentrations of up to 3.3 μM and 12.5 μM respectively, which were lower than those obtained in this research. For their part, ^{Landini et al. (2011)} used high I^-concentrations ranging from 5,000 to 20,000 μM in tomato plants grown hydroponically and noted that, despite toxicity symptoms such as chlorosis and leaf tip burn, the plants survived and produced fruits; therefore, they concluded that tomato plants are resistant to high iodine concentrations. Similarly, ^{Kiferle, Gonzali, Holwerda, Real-Ibaceta, and Perata (2013)} carried out an experiment in tomato plants applying iodine to the soil on a weekly basis. They found that at concentrations of 1,000, 2,000 and 5,000 μM I^-, and of 500, 1,000 and 2,000 μM IO₃^-, plant biomass did not decrease with respect to the control, with the exception of 5,000 μM I^- applications.

Other crop species such as lettuce grown hydroponically showed higher susceptibility to the presence of iodine, with reduced biomass being reported when I^- concentrations were equal to or higher than 40 μM, but without displaying any negative effects with the application of 240 μM IO₃^- (^{Blasco et al., 2008}).

Superoxide dismutase

SOD enzymatic activity decreased in half of the treatments compared to the control plants. The treatments that showed such behavior were biweekly I^- foliar application and I^- applied to the substrate, as well as daily IO₃^- and I^- foliar application (Table 1). These results are similar to those obtained in lettuce by ^{Blasco et al. (2011)} for treatments applied with 20, 40 and 80 μM I^-, which showed reduced SOD activity compared to the control plants. However, the same authors found that IO₃^- at 40 μM presented increases in SOD enzymatic activity. ^{Leyva et al. (2011)} obtained increased enzymatic activity after applying 80 μM IO₃^-. Furthermore, ^{Gupta et al. (2015)} found that this same activity increased in treatments with 40 μM IO₃^-, but not with 20 or 80 μM.

Superoxide anion is usually the first free radical formed naturally in photosynthesis and respiration; therefore, SOD represents the primary control line against oxidative stress (^{Gill & Tuteja, 2010}). However, in the present experiment the SOD activity of this enzyme was decreased. Although there are few reports in this regard about terrestrial plants, in marine plants it has been found that iodide is able to react quickly and directly with free radicals from biochemical energy systems, such as superoxide, singlet oxygen and even hydroxyl, at rates 12 to 500 times higher than ascorbate or glutathione (^{Küpper, et al, 2008}; ^{Luther, Wu, & Cullen, 1995}). There is a possibility exist that the same could happen in the case of tomato plants, resulting in direct neutralization of free radicals, thus, causing decreased SOD activity.

Catalase

In regard to catalase activity, no statistically significant differences were found between treated and control plants. This result differs to those obtained by other authors such as ^{Blasco et al. (2011)}, who observed increased enzymatic activity at concentrations higher than 80 μM for both I^- and IO₃^- treatments, finding severe toxicity damage and biomass reduction. ^{Gupta et al. (2015)} also reported increased catalase activity for IO₃^- treatments at 40 μM combined with 100 μM CdCl₂, possibly due to stress induced by the heavy metal. The function of catalase is to reduce peroxide to oxygen and water to prevent the formation of OH- radical (^{Asada, 1999}). It is likely that by decreasing SOD activity, H₂O₂ formation is also reduced while maintaining CAT activity at a basal level, which could explain the absence of differences.

Ascorbate peroxidase

Like CAT, APX showed no significant differences compared to control plants. This result contradicts those obtained in lettuce plants by ^{Blasco et al. (2011)}, who observed increased APX activity after applying IO₃^- concentrations of 20, 40 and 80 μM, without biomass reduction.

Meanwhile, ^{Gupta et al. (2015)} reported increased APX activity after applying 20 and 40 μM IO₃^- to plants treated with CdCl₂ at 100 μM. It is known that APX is involved in H₂O₂ detoxification, using ascorbate (AA) as the electron donor. In this study, the iodine treatments apparently did not alter the basal status of this enzyme, possibly due to the previously explained effect whereby SOD decreases and hence reduces H₂O₂ production.

Glutathione peroxidase

Similar to what was described above for APX and catalase, GPX activity showed no differences among treatments. The absence of differences among treatments is probably because this enzyme also degrades H₂O₂, which explains the decrease in SOD activity.

Glutathione

In the GSH results, an increase in two of the four biweekly IO₃^- and I^- treatments applied to the substrate were found, while the effect was considerable in the daily I^- foliar application treatment, which coincides with the decrease in SOD activity.

Although no information was found about the relationship between iodine and glutathione in tomato plants, ^{Leyva et al. (2011)} obtained an increased glutathione concentration after applying IO₃^- at 20 μM in lettuce plants grown hydroponically. The response, however, was different in the study carried out by ^{Blasco et al. (2011)}, who found decreased glutathione concentration when IO₃^- was applied at 80 μM in lettuce plants. This indicates that the influence of iodine varies according to the plant species, its chemical forms and applied concentrations, among other factors.

Ascorbate

Ascorbate concentration increased after daily foliar I^- application, the same treatment in which SOD activity decreased and GSH concentration increased (Table 1). Meanwhile, ^{Weng et al. (2008b)} reported an increase in ascorbate concentration in spinach grown hydroponically by adding I^- at 0.33 and 0.66 μM, but the same did not happen for IO₃^- . On the other hand, ^{Blasco et al. (2011)} reported the highest ascorbate concentration in lettuce plants for I^- treatments at 80 μM, the same concentration at which the plants showed a severe biomass decrease. Also, ^{Leyva et al. (2011)} found increased ascorbate in lettuce plants by applying IO₃^- at concentrations below 40 μM. The discrepancy in the results may be explained by the use of different plant species and chemical forms of iodine.

Total phenols

Total phenols showed no significant statistical differences between treatments and control. Furthermore, no other studies in which these compounds were quantified after applying iodine were found. However, part of the reason why iodine did not change the basal status of these compounds may be related to the fact that phenol accumulation is induced in response to increased H₂O₂ resulting from oxidative stress (^{Sakihama, Cohen, Grace, & Yamasaki, 2002}). Once again, the reduced SOD activity probably resulted from an antioxidant iodine effect, which could have been the reason for the absence of differences among treatments.