Plant material and growth conditions

The experiment was carried out in campus Montecillo, Colegio de Postgraduados, Montecillo, Estado de Mexico. The seedbeds for the seed germination and plantlet growth were established in a controlled environment chamber, with temperatures between 23 and 25 °C and a relative humidity of 50 %, with a light period of 14 h (fluorescent lamps) and 10 h of darkness. For the seedbeds, we used plastic trays with 50 cavities. Each tray was filled with the substrate peat-moss Sungro®; one seed was placed in each cavity and covered with a thin layer of vermiculite. The sweet pepper variety planted was Sidenca, of Enza Zaden company. Irrigation was carried out daily using a hand-held sprinkler to maintain the humidity of the substrate. Nutrition was provided wit Steiner solution at 25 % [0.147 mM Ca(NO₃)₂, 0.034 mM KH₂ PO₄, 0.076 mM K₂ SO₄, 0.176 mM KNO₃, 0.068 mM MgSO₄·7 H₂O, micronutrients, pH 5.5; it began once the first true leaves appeared, 7 d later, the concentration was increased to 50 % and 7 d later, to 75 % until transplant.

Foliar application of iodine

When seedlings presented the first true leaves, the foliar applications with iodine were performed, with intervals of 14 d. The treatments were 10, 15, 30, 45, and 50 μM KI L^-1 and a control (0 iodine), with three repetitions. Foliar applications were sprayed using a low-volume spray outside the growth chamber to avoid contamination between treatments. The experimental unit was a tray with 50 cavities, and the experimental design was completely randomized.

Measurement of variables during crop development

Samples were obtained 7 d after each foliar application of iodine, collecting 10 seedlings per experimental unit to measure: plan height (cm), from point zero on the surface of the substrate to the apex; stem diameter (mm), with a digital Truper brand vernier; foliar area (cm²), obtained by planimetry (scanning and analysis of images and estimation from the length and width of leaf), using the method reported by ^{Melgarejo et al. (2011)}. Seedlings were dried for 72 h in a continuous circulation oven at 70 °C and weighed to obtain dry weight in g.

Determining growth indixes

Data on dry matter (DM) and foliar area were used to determine growth indixes for sweet pepper: net assimilation rate (TAN= LnA₂-LnA₁/ t₂-t₁); absolute growth rate (TCA= P₂-P₁/t₂-t₁); relative growth rate (TCR= LnP₂-LnP₁/t₂-t₁); specific leaf area (AFE= AF/ leaf biomass). Ln A= natural foliar area logarithm (cm²); t= time (d); P= total dry weight (g); Ln P= natural logarithm of total DM (g) (^{Amaro et al., 2004}).

Determining total antioxidant activity (DPPH method)

The total antioxidant activity was determined using the DPPH method (^{Scherer and Texeira 2009}). The reaction is based on a reduction in color that takes place when the electron of a nitrogen atom in DPPH is reduced by receiving a hydrogen atom from the antioxidant compounds. In 10 repetitions per treatment, the plant material was softened. Once it was homogenized, 100 mg were weighed and placed in 2 mL microtubes. We added 1.5 mL of ethanol at 60 % and let it settle fro 24 h. Samples were centrifuged for 15 min at 9 000 rpm, 400 mL of the supernatant were taken from each sample and placed in a 2 mL microtube toadd600mLofmethanolat80%and1mLoftheDPPH solution. The samples were settled for 15 min and a reading was taken on the spectrophotometer at 517nm. The reading of each sample was repeated after 30 and 60 min. The results are expressed as a % of the DPPH inhibited.

Content of iodine and minerals content in seedlings

The iodine content was determined using the alkaline ash technique (^{Fisher et al. 1986}). The ten seedlings sampled for each treatment were placed in a continuous circulation oven at 70 °C for 72 h, ground, and 0.5 g of the dry plant tissue were weighed. The samples were placed in porcelain crucibles, KOH and KNO₃ were added, the crucibles were placed in a muffle at 580 °C for 3 h, they were then cooled at room temperature and the ashes were transferred to conical flasks for extraction with KOH. The ashes of the tubes were centrifuged at 7200 rpm for 15 min. Finally, 1 mL of the supernatant decanted and graduated at 10 mL with KOH 2M and readings were taken for quantification of iodine in the inductively coupled plasma optical emission spectrometry (ICP-OES, Varian 725-OES, Australia).

The contents of P, K, Ca, Mg, S, B, Cu, Fe, Mn, and Zn in the aerial section were analyzed using the humid digestion technique and read in the ICP, and N was determined using the Kjeldahl method (^{Bremner, 1965}).

Statistical analysis

The experimental design was completely randomized with six treatments and three repetitions per treatment. Data were analyzed using an analysis of variance and means comparison was carried out using Tukey test (p≤0.05).

Measuring crop development

The sweet pepper seedlings showed a significant response (p≤0.05) to the foliar iodine applications for plant height, stem diameter, foliar area, and DM for each treatment. Height increased with the foliar iodine application (10 μM L^-1), and it was different to the other the treatments (Table 1; p≤0.05). The results show that increasing the concentration of iodine reduced plant height and, at the highest concentrations, the edges of the leaves displayed symptoms of toxicity. The positive response in plant height could be due to an antioxidant effect of iodine, which would help the plant adapt to the growth environment. This antioxidant effect was mentioned by ^{Venturi (2011)}, although it depends on the concentration of iodine in the growth medium, since it is toxic at a higher concentration (^{Caffaggni et al., 2011}; ^{Landini, et al., 2011}). The same response was found in onion (Allium cepa L.), carrot (Daucus carota L.), celery (Apium graveolens L.), and spinach (Spinacea oleracea L.); with iodine applications higher than 5 μM L^-1, and with 40 μM L^-1 or more, plants were smaller and with symptoms of toxicity (^{Jiu et al., 2004}).

Stem diameter increased up to 32 % with respect to the foliar application of 10 μM L^-1 (Table 1), and 20 % with 45 μM L^-1. The thinnest stem was observed in seedlings with the highest iodine doses, along with burns on some leaf edges.

The foliar area was maintained in seedlings with iodine applications of medium concentrations (10 to 45 μM L^-1) (Table 1). The application of 50 μM L^-1 produced plants with smaller leaves and some symptoms of toxicity. The same trend is reported in the biofortification of lettuce (Lactuca sativa) with seven concentrations (10, 20, 30, 40, 80, 160, and 240 μM L^-1 KI), since there is a reduction in plant growth and biomass production with doses greater than 20 μM L^-1 of KI, and reduction is most notorious with 40 μM L^-1 KI. The toxic effect of iodine in high concentrations is described for various species (^{Caffaggni et al., 2011}; ^{Landini, et al., 2011}; ^{Zhu and Liu, 2003}), yet it is not properly understood. It is likely that the excess iodine interferes with the metabolism and cellular signaling of the ROS, causing negative responses on growth and other processes.

Dry matter was promoted when applying 10 μM L^-1 of iodine on the leaves; the control seedlings showed the lowest dry weight (Table 1), and with intermediate doses, the values were lower. ^{Jiang et al. (2001)} report similar results, as well as a phytotoxic effect on plant growth, and it may be due to an excessive accumulation of iodine in the plant tissues, or that once absorbed, intracellular oxidation to I₂ can occur, producing an inhibition of the photosynthetic process, which reflects on the growth of the plant.

Growth indices

^{Andrews et al. (2001)} and ^{Raven et al. (2005)} indicate that levels of radiation, photoperiod, water and nutrients have a direct influence on the accumulation of DM and foliar area, which are determining factors in growth indices. And in leaves carbohydrates are synthesized, which will be distributed to plant organs (^{Amaro et al., 2004}).

Applying iodine modified the dynamics of DM accumulation, changing the values of growth indices (Table 2). Data (Table 1) indicate that the 10 μM iodine treatment displayed the highest dry weight, but data shown in Table 2 indicate that the dynamic behavior of the accumulation was different in each case. According to these results, iodine application in different concentrations modify the strategy of the biomass distribution, its production efficiency and how it is used to create photosynthetic structures.

The TAN values in Table 2 indicate, for each treatment, the dry weight produced per unit of foliar area. Treatments with 10 and 15 μM produced less biomass per unit of foliar area, but they have the highest foliar area (Table 1) and accumulated a greater amount of dry biomass. The iodine treatments with 30, 45, and 50 μM were more effective than the control for biomass accumulation per unit of foliar area, and they accumulated more dry weight than the control. Changes in TAN indicate morphological, physiological, and biochemical modifications related to carbon metabolism (^{Shipley, 2002}). In marine plants, iodine has a significant effect on the photosynthetic metabolism (^{Kupper et al., 2008}), although we have no information available on this subject regarding terrestrial plants.

The results for TAC and TCR (Table 2) show a complex behavior towards iodine concentrations. The high TCA values in treatments with 30 and 45 μM, which did not have the highest final dry biomass (Table 1), show that seedlings did not grow uniformly, but rather had a combination of slow and rapid growth periods, which could identify conflicts between supply and demand. Nevertheless, they presented a greater biomass than the control. TCR was highest for treatments with 10, 15, and 30 μM, indicating that the highest efficiency in biomass accumulation took place with the intermediate iodine concentrations.

The data for AFE (Table 2) indicate the changes induced by the concentration of iodine in the strategy of the plant to distribute the dry biomass. AFE is an indicator of how the plant accumulates dry biomass in the foliar structures. The low AFE values in treatments with 10 and 15 μM show that plants had a high density of photosynthetic structures, evident due to the large amount of mass per unit of foliar area, as compared to control and the application of 30 and 45 μM KI. Potentially, this characteristic would relate to a higher efficiency of radiation absorption PAR per unit of foliar area. (^{Yao et al., 2016}).

It is difficult to explain the above results in depth without more biochemical, anatomic, and genomic information. However, the results show a direction for research about the effect of iodine on crop plants.

Total antioxidant activity

Iodine applications, as potassium iodide, increase antioxidant capacity of plants tissues. In Figure 1 it is shown that all KI concentrations increased up to 12 % DPPH inhibition, even with the highest KI concentrations, which caused reductions in plants height as well as some toxicity symptoms. This finding is similar to that observed in lettuce leaves treated with KI doses lower than 40 μM (^{Leyva et al., 2011}). In another experiment with lettuce, 10 to 40 μM does caused a clear increment of total antioxidant activity, mainly due to an increase in levels of ascorbic acid, in its reduced form, and this molecule has a high capacity to donate electrons and directly neutralize free radicals in enzymatic and non enzymatic reactions (^{Blasco, 2010}). The antioxidant response obtained with these treatments is relevant because it increases plants tolerance to stress conditions (^{Stevens et al., 2008}).

Figure 1 Antioxidant activity in sweet pepper seedlings with foliar application of six KI doses. 

Content of iodine and minerals in the aerial section of seedlings

The concentration of iodine in sweet pepper seedlings was directly proportional to the foliar dose applied (Figure 2). There was a considerable increase in iodine concentration in treatment with 45 μM L^-1, and threefold with 50 μM L^-1 with regard to the treatments.

Figure 2 Concentration of iodine in sweet pepper sedlings tissues with foliar application of six KI doses. 

The results of our study show that iodine absorption by sweet pepper seedlings is directly proportional to the concentrations applied and there is a similar trend for iodine in the aerial section and total antioxidants. These results are similar to those reported by ^{Ujowondu et al. (2010)} who biofortified different species of vegetables, native to Nigeria, with iodine and there was a higher concentration of iodine in plant tissues when the doses of potassium iodate or iodide increased in their treatments.

^{Jiu et al. (2006)} point out that iodine concentration increases in leaves and roots of spinach when increasing the doses of potassium iodate and iodide. Besides, ^{Zhu et al. (2003)} report that the accumulation of iodine in foliar tissue of spinach is directly proportional to doses of iodine supplied.

For the nutritional concentration of the aerial section, results show that the application of iodine increased nitrogen concentration by an average of 5 %, except with 45 μM L^-1, which reduced the amount of N, possible due to the effect of toxicity (Figure 3).

Figure 3 Concentration of total nitrogen in sweet pepper seedlings tissues with foliar application of six KI doses.  

The lowest concentration of P, K, Ca, Mg, and S (Cuadro 3), was found in control plants. This chart shows that applying iodine in low and medium doses increase the concentrations of the elements quantified. ^{Blasco et al. (2011)} indicate that applying iodine on lettuce in doses of no more than 40 μM L^-1 promotes nutritional quality without phytotoxic effects, and therefore iodine is considered a beneficial element for plants and can be used in these doses. ^{Jiu et al. (2004)}, in a study of the effect of residual iodine in the soil, mentions that low concentrations of this element promote absorption and the use of essential elements in an important number of plant species.

Results (Table 2) indicate that there is an optimum dose of iodine application on leaves, of 10 to 15 μM L^-1, since it promotes nutrient absorption. Besides, the results on the increase of foliar area by iodine absorption show that iodine promotes growth and minerals absorption in sweet pepper seedlings.