Iodine application affects nitrogen-use efficiency of lettuce plants (Lactuca sativa L.)

Abstract

The main objective of this work was to determine whether nitrogen-use efficiency was affected by the application of different forms (iodide vs iodate) and dosages (20, 40 and 80 µM) of iodine, to ascertain the influence of this trace element in a biofortification programme in lettuce plants. The parameters analysed were root and shoot biomass, nitrate concentration, and organic and total nitrogen as well as those defining nitrogen-use efficiency in plants: total nitrogen content, total nitrogen accumulation, nitrogen-uptake efficiency and nitrogen-utilization efficiency. In addition to decreasing shoot biomass, iodide treatments reduced leaf levels of nitrates, organic nitrogen, and total nitrogen content. Iodate treatments did not affect the concentration of nitrogen in its different forms. The application of iodide caused total nitrogen accumulation and nitrogen-uptake efficiency to decrease, iodate application improved the latter. Both iodide and iodate applications significantly improved nitrogen-utilization efficiency in comparison to the control. The results obtained show that iodate application rates of 40 µM or lower significantly improved all nitrogen parameters analysed, making it possible to increase lettuce productivity and quality.

Keywords:

• Biofortification
• iodine
• Lactuca sativa
• nitrate
• nitrogen-use efficiency

Introduction

Today, agriculture utilizes most of the fertile land worldwide, as well as a large proportion of water resources and fertilizer inputs. Furthermore, the population explosion has given rise to an increasing demand for plant food, since this is the main source of vitamins, minerals and biologically active compounds. This has raised the urgent need to increase yields without expanding the farming area (Lawlor, 2002; Kmiecik et al., 2004).

At present, plant utilization of trace elements such as iodine and selenium are under intense research, since these elements are essential for human health and are supplied through plant food. Iodine is regarded as an essential trace element for mammals, and its deficiency is related to numerous severe disorders such as goitre, reproductive failure, mental backwardness and brain damage. Today, about 30% of the world's population suffer from iodine deficiency (White & Broadley, 2005; Abrahams, 2006). To mitigate such nutritional disorders, biofortification has recently been proposed for improving plant food nutritional quality. Biofortification has been defined as the process of increasing the concentration of bioavailable essential elements (e.g. iodine, selenium, zinc, etc.) in the edible parts of crops through agronomic practices or genetic selection (White & Broadley, 2005). Although iodine-biofortification programmes have so far succeeded in promoting the intake of this element through plant consumption (Cao et al., 1994; Zhu et al., 2003; Blasco et al., 2008), none of the above works studied whether iodine biofortification influences basic plant physiological processes, even though there is evidence that an excessive application of iodine can cause phytotoxicity (Blasco et al., 2008). Essential processes for plants that can be altered by iodine biofortification are nitrogen (N) uptake, utilization, and accumulation, which in the case of lettuce cultivation, as we comment below, are determining factors in production and quality of this crop.

Nitrogen is often the most limiting factor in plant growth, which strongly depends on the amount of it available in the soil (Lea & Azevedo, 2006). The importance of the yield in agriculture, where N plays a decisive role, has led to a significant increase in the inefficient use of N fertilizers over the last decades, which has generated serious environmental contamination (Gastal & Lemaire, 2002; Hirel et al., 2007). Furthermore, leaf nitrate () accumulation can pose a problem for crops when both the application rate and uptake exceed plant growth needs (Ruiz & Romero, 1999; Prasad & Chetty, 2008).

The fact that anions are mainly accumulated in the leaves affects the nutritional quality of crops (Santamaría et al., 1999). Many species, such as lettuce, spinach, beets, radishes and celery, tend to accumulate large amounts of (MAFF, 1998). For instance, lettuce is the most grown and commercialized salad crop in the world (Abu-Rayyan et al., 2004), so its content is strictly regulated by law. represents a risk for human health, since when ingested it is rapidly transformed into nitrites and N-nitrous compounds, which can cause serious pathological disorders (Mensinga et al., 2003).

Apart from the environmental pollution caused by and the risks that its excess entails to human health, N fertilizers represent a financial cost to the farmer. Such disadvantages can be reduced either by (1) using different types of fertilizer (as well as other physical variables), which can improve nitrogen-use efficiency (NUE) utilization efficiency while lowering nitrate accumulation (Abu-Rayyan et al., 2004); or (2) growing cultivars with improved NUE, which, also has high commercial value, as it can increase yields with lower nitrogen application rates, even in soils where N is a limiting element (Svečnjak & Rengel, 2006).

The concept of NUE can, in turn, be divided into two other sub-parameters: (1) N-uptake efficiency (NUpE): the ability of the plant to absorb N from the soil, which is normally present as or ions; and (2) N- utilization efficiency (NUtE): the plant's capacity to translocate N to the shoots (Lea & Azevedo, 2006; Ruiz et al., 2006).

As mentioned above, many studies are focused on biofortification programmes using trace elements, including iodine, with the aim of boosting the intake of iodine in humans through plant consumption. However, very few of these studies have analysed the impact of this element on plant physiology. Therefore, the aim of the present work was to determine whether NUE and foliar concentration are affected by the application rates and forms of iodine.

Materials and methods

Plant material and growing conditions

Seeds of Lactuca sativa L. var. longifolia were germinated and grown for 35 days in cell flats of 3×3×10 cm filled with a perlite mixture substratum. The flats were placed on benches in an experimental greenhouse located in southern Spain (Granada, Motril, Saliplant S.L.). After 35 days, the young plants were transferred to a growth chamber under controlled environmental conditions, with relative humidity of 60–80%, day/night temperature of 25/15 °C, a photoperiod of 12 h at a photosynthetic photon flux density (PPFD) of 350 µmol m⁻² s⁻¹ (measured at the top of the seedlings with a 190 SB quantum sensor, LI-COR Inc., Lincoln, Nebraska, USA), and transplanted to individual 8-litre pots (25 cm upper diameter, 17 cm lower diameter, 25 cm height), filled with vermiculite as a substratum.

Throughout the experiment the plants were treated with a growth solution made up of 4 mM Ca(NO₃)₂, 6 mM KNO₃, 2 mM MgSO₄ 7 H₂O, 1 mM NaH₂PO₄ 2 H₂O, 50 µM H₃BO₃, 2 µM MnCl₂ 4 H₂O, 1 µM ZnSO₄ 7 H₂O, 0.1 µM Na₂MoO₄ 2 H₂O, 0.25 µM CuSO₄ 5 H₂O and 10 µM Fe-EDDHA. This solution, with a pH of 5.5–6.0, was changed every three days.

On day 45 after germination, the treatments were applied together with the above nutrient solution. The treatments consisted in the addition of I⁻ as potassium iodide (I⁻) (20, 40, 80 µmol iodine L⁻¹) and as potassium iodate () (20, 40, 80 µmol iodine L⁻¹) to the growth solution for 21 days. The different iodine application rates were selected on the basis that they had been shown to be the most appropriate for an iodine biofortification programme in lettuce (Blasco et al., 2008). In addition to these treatments, we included a control treatment consisting of applying the total growth solution, but without the iodine supplement.

The experimental design was a randomized complete block with 7 treatments, arranged in individual pots with six plants per treatment (one plant per pot) and three replications each. The experiment was repeated three times under the same conditions (for each treatment, we have 3 measurements per replicate and 3 replicates per experiment×3 experiments, n = 27).

Plant sampling

Lettuce roots and edible leaves (12–17 leaves per plant) were harvested 66 days after germination. Roots and leaf tissues were disinfected with 1% non-ionic detergent, rinsed three times in distilled water and dried with filter paper. These materials were used to quantify the volume of fresh biomass. Then, samples were lyophilized to determine dry mass and tissue total N, organic N and nitrate-N contents.

Plant analysis

For the determination of the iodine concentration, 25 mg of dry plant tissue was subjected to a mineralization process with 2.5 ml concentrated HNO₃ and 1 ml H₂O₂. The resulting solution was diluted in 25 ml distilled water and the concentration of the element was determined by atomic-absorption spectrophotometry following Soudek et al. (2006). The iodine concentration in the leaves was expressed as mg kg⁻¹ dry weight (DW).

The content was analysed on an aqueous extraction of 0.2 g of dried ground leaf material in 10 mL of Millipore-filtered water. A 100-µL aliquot was extracted and added to 10% (w/v) salicylic acid in 96% sulphuric acid. The concentration was measured by spectrophotometry (Cataldo et al., 1975) and the results were expressed as mg kg⁻¹ DW.

For the determination of organic N concentration, a sample of 0.1 g DW was digested with sulphuric acid and hydrogen peroxide (Wolf, 1982). After dilution in deionized water, a 1-mL aliquot of the digest was added to the reaction medium, containing a buffer solution (5% potassium sodium tartrate, 100 µM sodium phosphate, 5.4% (w/v) sodium hydroxide), 15% (w/v) sodium silicate, 0.03% (w/v) sodium nitroprusside and 5.35% (v/v) sodium hypochlorite. The samples were incubated at 37 °C for 15 min and organic N concentration was measured by spectrophotometry (Baethgen & Alley, 1989). Total N concentration (TNC) was assumed to represent the sum of organic N and concentrations. Organic and total N concentrations were expressed in mg N g⁻¹ DW.

Total N accumulation (TNA) was calculated by multiplying the TNC by the DW of total edible leaves (Sorgona et al., 2006), the results being expressed as mg N in edible leaves.

NUtE was calculated as the quotient between edible leaf DW and TNC (Siddiqi & Glass, 1981). The results were expressed in g² DW mg⁻¹ N.

1

NUpE was calculated as the quotient between the TNA and the root dry weight (RDW, Elliot & Läuchli, 1985), the results being expressed in mg N g⁻¹ DW

2

Statistical analysis

The data compiled were submitted to an analysis of variance (ANOVA) and the differences between the means were compared by Fisher's least-significant difference test (LSD). In addition, to ascertain whether the iodine application rate and forms used in the experiment significantly influenced the results, a two-way ANOVA was used and the means were compared by Fisher's LSD test. The significance levels of both analyses were expressed as: * p<0.05; ** p<0.01; *** p<0.001 and NS (not significant).

Results and discussion

As for biomass determination, it is one of the essential parameters in research on nutrient efficiency, especially in lettuce plants, where the growth of the aerial part is a determining factor in the agricultural value of this crop. Our research showed that the root biomass was not affected by the different I⁻ or treatments (Table I). With regard to the biomass of the edible parts, we found that I⁻ application reduced growth of the aerial part, which reached its lowest values with the 80 µM treatment (Table I). Conversely, application increased the biomass of the edible parts in all tests in comparison to the control treatment, the highest values being reached with an application rate of 20 µM (Table I).

Table I. Influence of the different application rates and forms of iodine on root and shoot biomass and foliar iodine and concentration.

	Root biomass		Shoot biomass		Iodine concentration		concentration
Treatments Iodine doses (µM)	I^−		I^−		I^−		I^−
0	0.47		3.02		Nd		89.2
20	0.35	0.40	2.85	3.75	844	566	58.0	81.6
40	0.37	0.39	2.98	3.55	935	614	56.7	88.2
80	0.38	0.38	2.34	3.37	1024	689	52.0	93.1
P-value	NS	NS	***	*	***	***	***	NS
LSD	0.14	0.11	0.21	0.35	21.38	85.27	4.89	10.9
Analysis of variance
Forms (F)	NS		***		***		***
Doses (D)	*		***		***		***
F×D	NS		***		***		***
LSD	0.12		0.26		40.41		5.53
Root and shoot biomass were expressed in g DW; Foliar iodine concentration was expressed in mg kg^−1 DW; Foliar concentration was expressed in mg kg^−1 DW. Nd: not detected.
The levels of significance were represented as follows: NS: (not significant), * p<0.05, ** p<0.01 and *** p<0.001.

Coinciding with our results, Mackowiak and Grossl (1999) found lower shoot biomass in rice with I⁻ concentrations of 10 µM or higher. On the other hand, these authors reported that a concentration of 100 µM decreased root biomass, whereas treatments showed no significant effect. Like in the previous case, Zhu et al. (2003), working with spinach plants, observed a direct relationship between the decrease in leaf biomass and the increase in I⁻ application rates to 10 and 100 µM. However, Dai et al. (2004) found that the concentration had a positive effect on the biomass of edible parts in pak chi and spinach.

In general iodine is not phloem mobile; on the contrary, especially in leafy vegetables, it is preferentially transported via xylem (Zhu et al., 2003). The phytotoxic effect of I⁻ on the growth of our plants may have been caused either by an excessive accumulation of this trace element in the plant tissues or by the intracellular oxidation to I₂ after the uptake, which can inhibit photosynthesis (Mynett & Wain, 1973). Our analysis of the concentration of iodine in the aerial part of our plants found that there are significant differences between the treatments. In the I⁻ and treatments, the foliar iodine concentration increased in parallel with the application rate in the nutrient solution; the maximum concentration appeared when we applied 80 µM (Table I). The iodine concentration in lettuce leaves treated with I⁻ was generally greater than in plants treated with (Table I). That could be because must be reduced to I⁻ before uptake, a process that requires energy. Therefore, iodine accumulation in plants may be limited by the reduction process itself (Zhu et al., 2003).

While such factors as temperature and light intensity affect both lettuce growth and quality, N is often the most limiting factor in plant growth (Abu-Rayyan et al., 2004; Niu et al., 2007). Consequently, the concentrations of and organic and total N are measured so as to determine whether it was necessary to apply N fertilizers, since such concentrations are closely related to the plant physiological requirements (Lawlor, 2002).

An excessive accumulation of can pose a critical problem for human nutrition in leaf crops such as lettuce, owing to which the maximum level allowed for commercial lettuce plants is 4500 mg kg⁻¹ fresh weight (FW) (Santamaría, 2006). We found that foliar concentration did not exceed this limit, regardless of the iodine treatments applied (Table I). As for the effect of the treatments, I⁻ application (≥ 40 µM) lowered the foliar levels (Table I), organic N concentration and TNC (Table II) in comparison with the control group. This could explain the loss in biomass observed after the application of high dosages of this form of iodine (Table I). Although more research is needed in this respect, the decline in the foliar concentration of , organic N and TNC by the application of I⁻ suggests that this trace element applied under this form at high rates affects the absorption process of , either because the specific transporters are altered or else because there is some type of antagonism between the two anions. On the contrary, the treatments did not significantly alter concentration (Table I) but did prompt an increase, though not significant, of the organic parameters N and TNC with respect to control, and this could be associated with maintenance or greater shoot biomass observed after the application of the different rates of (Tables I and II).

Table II. Influence of the different application rates and forms of iodine on constant NUE indicators in lettuce plants.

	Organic N		TNC		TNA		NUpE		NUtE
Treatments Iodine doses (µM)	I^−		I^−		I^−		I^−		I^−
0	36.5		125.8		380.4		798.3		0.024
20	33.3	38.5	91.3	120.2	260.4	451.2	737.3	1124.3	0.031	0.031
40	31.2	39.7	88.0	127.9	262.6	454.3	710.6	1174.4	0.038	0.027
80	29.7	40.5	81.8	133.7	192.0	450.3	519.4	1185.6	0.028	0.025
P-value	*	NS	***	NS	***	**	**	**	***	***
LSD	4.08	3.64	5.29	11.4	38.0	40.5	70.6	144.1	0.002	0.001
Analysis of variance
Forms (F)	***		***		***		***		*
Doses (D)	NS		***		**		*		***
F×D	*		***		***		***		*
LSD	2.51		4.10		14.6		69.3		0.001
Organic N and TNC were expressed in mg g^−1 DW; TNA was expressed in mg N; NUpE was expressed in mg N g^−1 DW; NUtE was expressed in g^2 DW mg^−1 N.
The levels of significance were represented as follows: NS: (not significant), * p<0.05, ** p<0.01 and *** p<0.001.

With the purpose of studying the effect of iodine application on N utilization, we experimentally tested parameters such as TNA, NUpE and NUtE (Table II), which are generally analysed in these kinds of research (Balingar et al., 1990; Ruiz & Romero, 1999; Ruiz et al., 2006; Sorgona et al., 2006).

The experiment revealed that whereas I⁻ application diminished the TNA, increased this parameter when using rates of 20 µM or higher. I⁻ application, moreover, decreased the NUpE, especially with a rate of 80 µM (Table II). Conversely, improved the absorption efficiency with all the application rates tested. With regard to the NUtE, both I⁻ and significantly improved it in comparison to control experiments (Table II).

After analysing the set of parameters tested in this work, we found that when applying a constant rate of N in the growing substratum (see plant material and growth conditions in Materials and methods section), the addition of I⁻ decreased the nitrogen uptake, concentration and leaf content. This reduced the biomass only with an application rate of 80 µM, possibly owing to the fact that lower doses boost the translocation and use of available .

Nevertheless, significantly increased all indicators related to NUE without increasing N concentration in the plant, which also reflects in higher biomass production with application rates of 40 µM or lower.

Both N uptake and utilization by crops are widely variable. So, it is essential to do agronomic and physiological research on plant response to N so as to get an understanding of N utilization that constitutes a powerful tool for the selection of efficient cultivars.

Considering that many programmes of biofortification with trace elements are being performed, it has become extremely important to study how the application of these elements affects plant physiology and, particularly, N utilization in leaf crops. As far as we know, the present work is the first one to have studied the effect of iodine application on N uptake, utilization and accumulation in lettuce, and its results, apart from being revealing, have practical implications. The results obtained show that application rates of 40 µM or lower significantly improved all N parameters analysed, making it possible to increase lettuce productivity and quality.

Acknowledgements

This work was financed by the PAI programme (Plan Andaluz de Investigación, Grupo de Investigación AGR161) and by a grant from the FPU of the Mininterio de Educación y Ciencia awarded to BBL.