Outcomes of foliar iodine application on growth, minerals and antioxidants in tomato plants under salt stress

The experiment was carried out in a chapel-type greenhouse with passive temperature control, located in the Horticulture Department of the Autonomous Agrarian University, Antonio Narro, Coahuila, Mexico, located at 25°21′12.8″ north latitude and 101°01′51.9″ west longitude. The experiment was conducted at 1,711 mslm with a temperature range of 20 °C and 35 °C, a relative humidity between 50% and 60%, 70% of natural irradiance and a 430 ppm of carbon dioxide (CO₂).

Tomato (Solanum lycopersicum L.) variety ‘Rio Fuego’ seedlings were used as plant material, which was transplanted 4 weeks after sowing, on 10 May 2019, in 10 kg capacity polystyrene containers. Peat moss + perlite was used as a substrate in a 1:1 ratio.

Fertilisation was started 1 day after transplantation. Steiner (1961) nutrient solution was applied daily through an automatised system in irrigations of 200 mL for 5 min, five times a day. The fertilisers used and their concentrations were the following: Ca(NO₃)₂·4H₂O – 4.5 mmol, KH₂PO₄ – 1 mmol, MgSO₄·7H₂O₄ – 2 mmol, KNO₃ – 3 mmol, K₂SO₄ – 1.5 mmol, Fe chelated – 3 mg · L⁻¹, H₃BO₃ – 0.5 mg · L⁻¹, MnSO₄ – 0.7 mg · L⁻¹, ZnSO₄ – 0.09 mg · L⁻¹ and CuSO₄ – 0.02 mg · L⁻¹. The nutrient solution was maintained at pH 6.3 and an electrical conductivity (EC) of 1.8 dS · m⁻¹.

Iodine application was started 10 days after transplantation (DAT) using KIO₃. A total of three foliar treatments were carried out: (1) absolute control (SN), (2) saline control (NaCl at 100 mM) and (3) plants subjected to saline stress + iodate at 100 μM (NaCl + KIO₃). A total of 35 plants were used in each treatment, giving rise to a cumulative figure of 105 plants (Table 1).

The foliar applications were in both adaxial and abaxial sides of the leaves at a rate of 16 mL per plant or 400 L · ha⁻¹; every plant received 39 mg of iodine per litre, 20 dat (30 May 2019) and the stress treatment was started by adding NaCl as nutrient solution at 100 mM, through a completely randomised experimental design.

Three randomised samplings were carried out for the quantification of growth; the first one was carried out at 41 dat, in seedling stage; the second one at 71 dat, in the flowering stage; and the third sampling at 125 dat, in full production. In each sampling, five plants were taken, and were evaluated to assess the number of leaflets, counting each leaf blade within the compound leaves; the height of the plant was measured with a flexible tape from the base of the stem to the apex; the dry weight (DW) was obtained after placing the samples in an oven for 76 h at a temperature of 70 °C.

The K, Ca, Mg, Mn, Fe, Zn and Cu were mineralised by acid digestion as follows; 1 g of dry tissue was weighted in OHAUS analytical balance after 30 mL of nitric acid was added, and then they were placed on a heating plate until the clarification of the sample. Finally, it was made up to 100 mL with deionised water and filtered on Whatman #1 paper (Helrich, 1990). The sample was analysed using a Varian spectra fs-240 Atomic Absorption Spectrophotometer (AAS).

Total nitrogen was determined using the micro Kjeldhal technique (Muller, 1961), which was carried out by weighing 50 mg of dehydrated tissue, and placing it in a flask + 3 mL of digester mixture; once it had turned to a transparent green colour, the sample was transferred to the distiller tube adding 25 mL of sodium hydroxide to 50%; after this, 30 mL of boric acid + four drops of mixed indicator + distillation were placed in a beaker, until 60 mL of solution with a blue-green colour had been obtained. Next, the titration was carried out by placing a burette with 0.025 N sulphuric acid and dropping it into the distilled sample until a pink colour was obtained. Finally, the determination of the N content was made based on the spent volume (mL) of sulphuric acid.

Total phosphorous was determined by the spectrophotometric technique of aminonaphtholsulfonic acid (ANSA), according to the method suggested by Peterson (1978). First, 1 mL of the digested sample was taken and placed in a test tube along with 5 mL of an ammonium molybdate solution + 2 mL of ANSA solution; once stirred, it was allowed to stand for 20 min; the reading was carried out in a UV-VIS Thermo Genesys 102 spectrophotometer at 650 nm.

Iodine was extracted by alkaline ash technique (Ujowundu et al. 2009); 1 g of dry plant tissue was weighed and placed in crucibles; later 2 mL of 2 M KOH, and 1 mL of 2 M KNO₃ was placed in an oven at 100 °C for 2 h. Subsequently, the crucibles were put in a muffle at 580 °C for 3 h. Finally, the iodine was extracted with 2 mL of 2 mM KOH. The quantification was carried out using an inductively coupled plasma-optical emission spectrometry (ICP-OES) device, Agilent 725. The results were expressed in milligram of iodine per kilogram of DW.

Every fruit produced by the plants was counted and weighed in each treatment until the end of the cycle, and the total fresh weight was expressed in grams.

The lyophilised plant tissue was macerated and weighted at 100 mg. It was then transferred to centrifuge tubes with 10 mg of polyvinyl pyrrolidone; 2 mL of 0.1 M of phosphate buffer pH 7.2 was added and thereafter sonication was carried out for 10 min. Subsequently, the samples were subjected to microcentrifugation at 12,000 rpm for 10 min at 4 °C, and the supernatant was collected and filtered with a nylon syringe filter (Ramos et al., 2010). Finally, it was diluted 1:15 with phosphate buffer.

Assessment of CA was carried out by 2,2-diphenyl-1-picrylhydrazyl (DPPH) technique, wherein 50 μL of the biomolecule extract plus 50 μL of DPPH at concentration of 0.5 mM solution were added; they reacted for 15 min, and the reading was reckoned at 530 nm in a plate reader Biotek Exl 8000 (Mareček et al., 2017).

Lycopene was quantified spectrophotometrically according to the method suggested by Bunghez et al. (2011); 100 mg of dried tomato tissue was weighed and placed in 2 mL centrifuge tubes. After 1.5 mL hexane was added to the above, they were homogenised in vortex for 30 s, sonicated for 5 min and centrifuged at 4 °C for 10 min at 10,000 rpm. The supernatant was extracted and filtrated with syringe filters and read at 472 nm in ultraviolet visible spectra (UV-VIS) using Genesys 10S Thermo Scientific, Santa Clara, California, USA.

For the extraction of ascorbic acid (AsA), 100 mg of the lyophilised leaves were weighed, and 1 mL of water : acetone solution was added in a 1:1 ratio (Yu and Dahlgren, 2000). It was vortexed for 30 s, followed by sonication for 5 min. Finally, it was centrifuged at 4 °C at 12,500 rpm for 10 min, and the supernatant was extracted and filtered. Quantification was carried out using high-performance liquid chromatography (HPLC), for which a Thermo Spectra system P4000^® was used, under the following conditions: wavelength 230 nm, mobile phase NaH₂PO₄ 50 mM, pH 2.8, flow 1 mL · min⁻¹. Further, aquasil C-18 column was used at a temperature of 60 °C. Units were reported in grams per kilogram.

Glutathione was quantified following the spectrophotometric technique using 5,5 dithio-bis-2 nitro benzoic acid (DTNB) (Xue et al., 2001); 0.48 mL of the extract, 2.2 mL of dibasic sodium phosphate (Na₂HPO₄ at 0.32 M) and 0.32 mL of DTNB at 1 mM were placed in a centrifuge tube. It was mixed and read on a UV-VIS spectrophotometer at 412 nm. The results were expressed in grams per kilogram.

For the quantification of the total proteins, 100 μL of the biomolecule extract plus 1 mL of the Bradford reagent were added; the result was mixed and reacted for 2 min. Finally, it was read at 595 nm in the UV-VIS spectrophotometer, using bovine serum protein as a standard (Bradford, 1976).

Hundred milligrams of lyophilised leaves plus 1 mL of water-acetone solution was centrifuged at 10,000 rpm at 4 °C for 10 min, and the supernatant was obtained. Next, 50 μL of supernatant was taken, and 200 μL of Folin–Ciocalteu reagent plus 500 μL of 20% Na₂CO₃ and 5 mL of distilled water were added. Subsequently, it was incubated at 45 °C for 30 min. The samples were read with the spectrophotometer at 750 nm (Urias-Lugo et al., 2015).

Testing for catalase was carried out by measuring two reaction times, time zero (T0) and reaction time 1 min (T1). For T1, it was prepared by adding 0.1 mL of biomolecule extract plus 1 mL of H₂O₂, and after 1 min of reaction, 0.4 mL of 5% H₂SO₄ was applied. The substrate concentration (H₂O₂) was read at 270 nm in a spectrophotometer (Cansev et al., 2011).

Assessment for glutathione peroxidase was carried out with the method established by Xue et al. (2001) using H₂O₂ as a substrate. First, 0.2 mL of the biomolecules extract was placed in a test tube, with the addition of 0.4 mL of reduced glutathione (0.1 M) and 0.2 mL of Na₂HPO₄ (67 mM). Subsequently, 0.2 mL of 1.3 mM H₂O₂ was added to start the catalytic reaction. After 10 min, 1 mL of trichloroacetic acid (1%) was added to stop the reaction. This mixture was placed in an ice bath for 30 min. Then it was centrifuged at 3,000 rpm for 10 min. Next, 0.48 mL of supernatant was placed in a test tube, and then 2.2 mL of Na₂HPO₄ (0.32 M) and 0.32 mL DTNB were added. Readings were taken on the UV-VIS spectrophotometer at 412 nm.

The APX enzymatic activity was carried out according to the method of Nakano and Asada (1987); 100 μL of biomolecules extract, 500 μL of ascorbate (10 mg · L⁻¹) and 1 mL of H₂O₂ (100 mM) were added in a microcentrifuge tube. AsA was measured at 266 nm in a spectrophotometer (Thermo Scientific Genesys 10S UV-VIS) for T0, after 10 min (T1). The activity units (IU) were expressed in mM of ascorbate · min⁻¹ / total proteins.

The determination of the enzymatic activity of SOD was carried out using the SOD Cayman 706002® kit. A mixture of 20 μL of extract, 200 μL of radical detector (tetrazolium salt) and 20 μL of xanthine oxidase solution was placed in a microplate, shaken for 10 s and then incubated at 26 °C for 30 min. The absorbance was measured at 450 nm using a plate reader. SOD activity was expressed as percentage inhibition rate.

The experimental design was completely randomised with three treatments and 35 repetitions per treatment, resulting in a total of 105 plants for the experiment; each plant was considered as an experimental unit.

The data was analysed in univariate form, using a one-way analysis of variance (ANOVA) with five repetitions per treatment, followed by a least significant difference post-hoc test (LSD, p ≤ 0.05) using the Infostat software package (2018 version).

As shown in Figure 1, the highest amount of biomass was found in the absolute control plants, in the three samplings. On the other hand, the saline control (NaCl) and treated-with-KIO₃ plants (NaCl + KIO₃) did not show statistical differences, indicating that iodine applications did not prevent the biomass loss.

Figure 1

However, although iodine did not result in avoiding the loss of plant biomass, it improved yield (raising it by 23%), and resulted in a more significant number of fruits per plant, as indicated in Figure 2.

Figure 2

Table 2 shows other growth variables, such as fresh weight, height, number of leaflets and stem diameter, but no differences were found between salt control (NaCl), and KIO₃-treated plants.

As shown in Figure 3, the highest concentration of iodine was found in the leaves of the plants treated with KIO₃ (KIO₃ + NaCl) compared to NaCl control, with an iodine average of 9.25 mg · kg⁻¹. In the fruit, the range was between 1.3 mg · kg⁻¹ and 1.6 mg · kg⁻¹ D.W., with no statistical differences between treatments.

Figure 3

Table 7 shows the results obtained for enzymatic and non-enzymatic antioxidants, CA and total proteins found in tomato fruits treated with iodine and subjected to salinity stress. The most notable result was an increase in the content of AsA and total proteins in the plants treated with iodine and subjected to salinity stress; in contrast, it was found that the same treatments were characterised by a reduction in CA compared to NaCl control. In addition, an increase in glutathione peroxidase (GPX) activity was also observed in plants subjected to NaCl and KIO₃ + NaCl application. Finally, a decrease in lycopene was found with the iodine + NaCl treatment compared to controls.