TUNING

DESCRIPTION

TECHNICAL FIELD

The present invention relates to a method to control the enrichment of plants and particularly wheat plants in organic selenium, particularly selenomethionie (SeMet), selenocysteine (SeCyst) or a mixture of both, wherein the plant is fed with a nutrient solution enriched with at least one of sodium selenite and sodium selenate at a controlled molar ratio that produces corresponding SeMet/SeCyst ratio, leading thus to a selective selenoaminoacid production by varying the indicated Selenite/Selenate molar ratio (chemical tunning), regulation that can be expressed in terms of the related redox potential of the nutritional solution to produce enriched plants in organic selenium using this method, and to products obtained from this cereal plant.

BACKGROUND ART

Selenium (Se) is an essential trace element for animals mainly due to its presence in several vital enzymes, such as glutathione peroxidase (Deagen, Beilstein and Whanger 1991 ; Deagen et al. 1993; Polatajko, Jakubowski and Szpunar 2006). Its biological importance and its putative anticancer activity (Clark et al. 1996) or cardiovascular preventive action (S. Stranges et al, 2006) have resulted in the popularity of food supplements, usually based on selenite, selenomethionine or selenized yeast (Navarro-Alarcon and Cabrera-Vique 2008; Rayman 2004; Schrauzer 2001). The narrow margin between toxicity, essentiality and deficiency grows up the interest in Se because of both health and environmental impacts as relatively high Se concentrations in soils or waters represent a threat for the environment (Pyrzynska 2002).

The most important human source of Se is food; therefore plants play an important role in Se supplementation (Finley 2005). Plants synthesize selenoproteins from Se containing soils; hence Se content of crops depends on quality of soil Se richness. It is therefore important to better understand Se bioassimilation processes, changes in growth parameters and nutrient uptake in correlation with Se uptake and translocation in plants.

Cereal grains are poor sources of key mineral nutrients. As a result, the world's poorest people, generally those subsisting on a monotonous cereal diet, are also those most vulnerable to mineral deficiency diseases. We selected wheat in our study because this cereal and its products, for instance, breads, cakes, cereals and pasta, are an important source of Se intake for human diet (Lyons, Stangoulis and Graham 2003; Hawkesford and Zhao 2007). Wheat is a non- Se-accumulator plant and the threshold Se-toxicity concentration is dependent on the form of Se accumulated (Terry et al. 2000). On the other hand, Se concentration in wheat shows great variations between countries and regions (Hawkesford and Zhao 2007; Zhu et al. 2009). In this concern, the information about Se speciation in enriched wheat will be of key importance to understand the role of this element on the observed health benefits. Nowadays, it is well- recognized that the particular physico-chemical form of an element present in a sample will determine the toxicity, the biological activity, the bioavailability and the environmental impact of this element (Polatajko et al. 2006). To date, the mechanism of selenate uptake by plants has been already reported, however little is known of selenite related behaviour (Arvy 1993; Ellis and Salt 2003; Hopper and Parker 1999; Li, McGrath and Zhao 2007).

With respect to Se determination in plant samples and hydroponic solutions, ICP-MS has been applied as one of the most powerful tools for trace level concentration analysis in environmental and biological samples. Due to the interference of Ar dimmer ions in Se determination, we have used an ICP-MS instrument equipped with a collision cell, thus been able to remove spectral interferences by pressurizing the cell chamber with inert or reacting gases (Tanner, Baranov and Bandura 2002), i.e., the use of H2 in the case of our study. The approach for total Se determination in biological samples by ICP-MS with H2 gas in collision cells has been already reported (Huerta et al. 2003; Montes-Bayon et al. 2006; Wilburn et al. 2004).

On the other hand, there is a lack of knowledge with respect to the influence of the physicochemical parameters of the cultivation media on the finally produced selenoaminoacids by the enriched plant. A proper solution of this gap of knowledge will contribute to overcome two main important issues in organic selenium enrichment: 1) the variation of organic selenium quality due to both selenium content in the cultivation media and seasonality of such production. 2) to determine production conditions for a selected quality of organic selenium, i.e, specific relationship SeMet/SeCyst for a given application or disease prevention.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a method to control the enrichment of plants and particularly wheat plants in organic selenium, particularly selenomethionie (SeMet), selenocysteine (SeCyst) or a mixture of both, wherein the plant is fed with a nutrient solution enriched with at least one of selenium salt, such as sodium selenite and sodium selenate at a controlled molar ratio that produces corresponding SeMet/SeCyst ratio, leading thus to a selective selenoaminoacid production by varying the indicated Selenite/Selenate molar ratio (chemical tunning), regulation that can be expressed in terms of the related redox potential of the nutritional solution to produce enriched plants in organic selenium using this method, and to products obtained from this cereal plant.

In this work, the inventors have carried out an enrichment of wheat plants with Se supplied in the form of selenium salts, such as sodium selenate, sodium selenite or a mixture of both.

One of the objectives of the present study includes not only the assimilation of Se but also its effect on the micro and macronutrient uptake in each part of the plant (roots and shoots). In addition, we study effects of Se supplementation on growth parameters. Although, there are some studies about the effect of Se on the uptake of other essential elements in plants, the interaction between Se and essential nutrients is not still well understood (Arvy et al. 1995; Fargasova et al. 2006; Feng et al. 2009).

In its first embodiment, the invention is directed to a method to enrich plants in organic selenium, particularly in selenomethionine, selenocysteine or a mixture of both, wherein the cereal plant is fed with a nutrient solution comprising at least one selenium salt at a total selenium concentration less than 100μΜ having a redox potential between -0.2 and 0.95 volts (minus 0.2 and plus 0.95 volts) and a pH varying between 5.0 and 9.0.

In a second aspect, the invention is directed to a method to enrich plants in organic selenium, particularly in selenomethionine, selenocysteine or a mixture of both, wherein the plant is fed with a nutrient solution comprising at least one of sodium selenite (Na2Se03'5H20) and sodium selenate (Na2Se0 ₄ ) at a total selenium concentration less than 100μΜ having a redox potential between -0.2 and 0.95 volts (minus 0.2 and plus 0.95 volts) and a pH varying between 5.0 and 9.0.

In a third aspect, the invention is directed to cereal plants, particularly wheat plants, enriched in organic selenium using the method of the invention.

In a fourth aspect, the invention is directed to cereal products obtained from the cereal plants enriched in organic selenium using the method of the invention.

The invention is based on the fact that the inventors have found that, when a cereal plant such as a wheat is fed with a nutrient solution comprising at least one of sodium selenite (Na2Se03-5H20) and sodium selenate (Na2Se0 ₄ ) having a total selenium concentration less than 100 μΜ having a redox potential between -0.2 and +0.95 volts, the plant is enriched in organic selenium, particularly selenomethionine, selenocysteine or a mixture of both. The enrichment is more accused in the plant shoots rather than in the roots. The inventors have also found out a relationship between the redox conditions of the nutrient solution comprising the selenite and/or selenate and the predominant selenoaminoacid obtained. This finding provides a way to chemically tune the conditions for specific selenoaminoacids production. This aspect is based on the obtained results of this invention that reveal that the variation of Selenite/Selenate ratio produces a significative effect on the nature of aminoacids obtained. Additionally, according to this invention it is possible to observe how the production of Selenomethionine increases in favor of Selenocysteine when the Selenite/Selenate ratio decreases. This effect correlates the redox conditions of the hydroponic solution provided by the Selenite/Selenate ratio with the observed redox behavior of the corresponding selenoaminoacids Selenocysteine and Selenomethionine observed at the XANES Spectra (Synchrotron technique) of this invention. This finding provides us a way to tune chemically the conditions for a specific selenoaminoacid production.

In one embodiment of the method to enrich cereal plants in organic selenium, the method comprises feeding the plant with a nutrient solution comprising about 10μΜ selenium as sodium selenite to obtain a higher proportion of selenocysteine than selenomethionine in the cereal shoots. In other embodiment, the method comprises feeding the plant with a nutrient solution comprising about 10μΜ selenium as sodium selenate to obtain a higher proportion of selenomethionine than selenocysteine in the cereal shoots.

In another embodiment, the method comprises feeding the plant with a nutrient solution enriched with about 5μΜ selenium as sodium selenite and 5μΜ selenium as sodium selenate.

In an embodiment of the cereal plant enriched in organic selenium of the invention, the cereal plant can be wheat, maize, rice, barley, oat or sorghum. In an embodiment of the cereal product obtained from a cereal plant enriched with the method of the invention, the cereal product can be cereal grains, cereal flour or cereal stems

Brief description of the figures

Fig. 1. Selenium reference compounds. (REFERENCES SPECTRA): shows the Se K near- edge spectra for pure selenium reference compounds of SeCy, SeMet, selenite and selenate. As expected, a shift position of the edge features to higher energies as Se becomes more oxidized. The obtained spectra of SeMet and SeMeSeCys (data not shown) are very similar mainly due to the similar local structure around selenium in both amino acids (H3CSeCH2R). The same similarity is reported by Thavarajah et.al. in a study of selenium speciation in lentils (Thavarajah, D; Vandenberg A.; Graham, N.G.; Pickering I.J. Chemical form of selenium in naturally selenium- rich lentils (Lens culinaris L.) from Saskatchewan. J. Agric. Food Chem. 2007, 55, 7337-7341.). Principal component analysis results indicated that the set of spectra from each sample require four main components. Therefore, in the present study organic selenium is modeled as SeMet and SeCy and inorganic selenium as selenate and selenite, all considered the major selenium forms present in wheat hydroponically enriched samples.

Fig. 2. (CRM FIT SPECTRA): Shows a comparison between the Se K-edge spectrum of SeMet and the certified reference material SELM-1. Applying the same quantitative data analysis procedure as for wheat samples, the compositional result for SeMet content in SELM-1 is 100% with a residual value corresponding to the quality of the fit of 0.07. Thus, the result is in agreement with the selenium composition of SELM-1 and the accuracy and sensitivity of the method can be considered evaluated. Figure 3. (SYNTHETIC SAMPLES SPECTRA): Shows quantitative selenium speciation on Table 1 (Synthetic samples results table), the experimental fit and quantitative results are in accordance with the theoretical values. Synthetic samples containing the selenium studied species were analyzed to evaluate the performance of the developed analytical method. Additionally, multiple scans of each sample were performed to confirm the measurement reproducibility and check possible oxidation state alterations or species degradation during the analysis. All the analyzed samples do not show edge shifts or changes in the replicated spectra during the measuring time. Therefore, it is proved that the chemical state of the samples remained unchanged during the measurements.

Table 1 shows linear combination fit results of synthetic samples A and B. Theoretical and experimental composition raw quantitative results in percentage, residual values corresponding to the quality of the fit and sums of components for each sample.

Selenium Theoretical Experimental

Residual Sum (%)

components composition (%) composition (%)

SeMet rich synthetic sample (Fig. Xa)

SeOs* 10 7.0

SeCy rich synthetic sample (Fig. Xb)

SeOs* 10 7.2

SeCy 60 59.5

Table 1

EXAMPLES

The following examples illustrate the invention and should not be considered in a limitative sense thereof. The following examples have been carried out with following materials and methods:

Plant material and culture conditions Triticum aestivum cv. Pinzon (Semillas Fito S.A., Spain) was used in this study as soft wheat well-known for its high flour quality. Before starting the hydroponics culture, seeds were germinated on moist filter paper for 3 days in an incubator at 25°C. Seedlings were then transferred to 1 L plastic pots and were allowed to acclimate and grow for a week with ¼ strength Hoagland's nutrient solution. Afterwards plants were exposed to 5 days-long under different Se supplementation treatments.

The composition of the nutrient solution was: 1.0 mM KN03, 1.0 mM Ca(N03)2, 0.25 mM MgS04, 0.5 mM K2HP04, 2.0 μΜ MnCI2, 3 μΜ H3B03, 0.1 μΜ (NH4)6Mo7024, 2μΜ ZnS04, 1 μΜ CuS04 and 60 μΜ FeNa-EDTA. The pH of this solution was buffered at 6.0 with 2 mM MES (2-morpholinoethanesulphonic acid, pH adjusted with KOH). The solution was aerated continuously and renewed twice in the middle of growth period. The culture was carried out in a controlled culture chamber with the following conditions: a photoperiod of 16 h day/8 h night, a temperature of 24°C day/18°C night and a light intensity of 320 μΕ m-2 s-1.

The relative availability of selenate and selenite in nutrient solutions depend on the presence of other competing ions (Hopper and Parker, 1999). Higher accumulation of Se in shoots in selenate enrichments compared with that of selenite is in agreement with results reported by Ellis and Salt (2003) where it was proposed that selenate readily competes with sulfate for uptake by plants and is probably assimilated actively by sulfur transport pathway in chloroplasts due to their chemical analogy. In our study, selenite in nutrient solution at pH 6.0 exists primarily as HSe03- (Geochem, data not shown) and behaves as a weak acid that can compete with phosphate uptake. Other authors have proved that selenite uptake has a great passive component and a minor active one (Ulrich and Shrift, 1968; Broyer et al. 1972; Arvy, 1993; Hopper and Parker, 1999). The nutrient solution in most studies contains sulfate levels similar to those found in soil solution but phosphate concentrations are normally much higher (Asher and Edwards, 1983). On the other hand, Hopper and Parker (1999) demonstrated that the inhibition of sulfate by selenate is stronger than that of phosphate by selenite. Taking into account these findings in our experiments, levels of both nutrients have been lowered to avoid part of this competition, even though the selectivity of transporters varies between plant species and nutritional status (White et al. 2004).

Selenium enrichment The enrichment treatments were done by adding Se to the nutrient solution cultures in the form of sodium selenite (Na2Se03-5H20, Fluka) and sodium selenate (Na2Se0 ₄ , Fluka) separately at four different Se concentrations: 1 μΜ, 10μΜ, 50μΜ and 100μΜ. Additionally, the same Se total concentrations levels were reached by mixing both Se species at equal quantities. These treatments with mixed species are abbreviated as 0.5+0.5, 5+5, 25+25 and 50+50.

Each plastic pot had three plants and each Se treatment was duplicated. It is noteworthy that plant roots and shoots, separately, from each pot were mixed together before digestion. In addition, the complete hydroponics culture experiment was done twice.

Sample preparation

After 5 days of incubation in Se enriched media, plants were harvested and roots desorbed with CaCl2 solution to remove Se in root apoplast. After desorption, plants were rinsed with distilled water, divided into shoots and roots, frozen into liquid nitrogen and then lyophilised. Plants were weighted before and after lyophilisation in order to determine moisture content. Aliquots of nutrient solutions were taken for analysis of total Se and root elongation were measured to study the relative plant growth before and after Se enrichment.

Wheat shoots and roots samples were digested in a microwave oven (Mars 5, CEM, USA) equipped with HP500 PFA vessels (CEM, USA) with 2 mL of 70% HN0 ₃ and 2 mL of MilliQ water under the EPA 3052 microwave oven method. The obtained digested solution was filtered, properly diluted with MilliQ water and analysed to determine total Se concentration and other elements.

Total Se determination

Corresponding samples were analysed in an ICP-MS (PQExCell, Thermo Elemental, UK) in order to determine total Se concentration in each plant part after microwave acid digestion. A standard solution of 1000 mg L-1 of Se purchased from Aldrich (USA) was used for the calibration procedure and further dilutions were made with Milli-Q water. A flow of 3 mL min-1 of hydrogen was applied to pressurize the collision cell to be able to monitor 80Se and 82Se (B'Hymer and Caruso 2006). All Se isotopes were measured (76, 77, 78, 80, 82 and 83) and the effect of Se was quantified and corrected by measuring a known solution of Br (79Br and 81 Br). In the absence of wheat Se enriched reference materials available, SELM-1 CRM (Selenium Enriched Yeast Certified Reference Material, NRC, Canada) was analysed to test and validate the analytical methodology for total Se determination by ICP-MS. The SELM-1 CRM Se content analysed was 1980 ± 41 mg/kg (expressed as average ± SD, n=3). That result is in reasonable agreement with the values determined by NRC during the production of this material (2059 ± 64 mg/kg) (Mester et al. 2006).

Nutrient elements determination

Wheat shoots and roots digested samples were analysed by ICP-OES (Optima 3200RL, Perkin Elmer, USA) to determine the concentration of macronutrients (Ca, K, Mg, P and S) and several micronutrients (Cu, Fe, Mn and Zn) in order to study the effect of Se addition on these elements uptake. Corresponding standard 1000 mg L-1 solutions purchased from High Purity Standards (USA) were used for the calibration procedure.

Data analysis

Statistical analysis for comparison of means between different treatments was done by a two sample t-test at a significant level of 0.05 with SPSS software package. All results are expressed as means of 4 replicates with corresponding standard errors.

X-Ray Absorption Spectroscopy. The XANES experiments were carried out at the beamline C of HASYLAB (Hamburg, Germany) running at 4.45GeV with electron currents up to 120mA. The incident beam desired energy is selected by a fixed exit Si double crystal monochromator. The photon absorption of Se was recorded at the edge energy for its K line at 12658eV, and its Ka1 11224eV and Ka2 12497eV fluorescent line intensities were measured in fluorescence mode. All XANES spectra were collected at room temperature. The selection of the detection mode depends upon the sample concentration and the matrix background (Koningsberger, D.C.; Prins, R. X-ray absorption. Wiley, New York, 1988). Therefore, pure reference compounds diluted in cellulose were analyzed in transmittance mode, while fluorescence detection mode was used for the analysis of Se diluted wheat root and shoot samples.

The XAS data were averaged (3-5 scans), normalized and background subtracted using Sixpack software package (Webb, S. M. A graphical user interface for XAS analysis using IFEFFIT. Phys. Scr. 2005, T115, 1011-1014). Quantitative selenium speciation data were obtained by principal component analysis and linear least-squares fitting of the spectra from reference compounds, including sodium selenite (Na2Se03-5H20, Fluka) and sodium selenate (Na2Se0 ₄ , Fluka) as inorganic Se compounds, whilst Selenomethionine (SeMet, Across Organics) and Selenocystine (SeCy, Sigma Aldrich) were modeled as organic selenoaminoacids. Nowadays, the least-squares fitting is widely accepted as a valid method for the speciation of complex biological samples (Pickering, I.J.; Brown, G.E., Jr.; Tokunaga, T.K. Quantitative speciation of selenium in soils using X-ray absorption spectroscopy. Environ. Sci. Technol. 1995, 20, 610-613). To examine the sample composition as a linear combination of standard components various fittings of Se reference compounds were calculated repeating the process until no more significant components could be identified and the sum of all components was equal to 100% (±15%). The relative quality of the fit was quantified by the residual value, a measure of how close the fit is to the data based on a sum of squares determination of the fractional misfit defined as follows:

Additionally, fitting procedure accuracy and sensitivity were evaluated by the analysis of synthetic samples and the certified reference material SELM-1 (Selenium Enriched Yeast Certified Reference Material, NRC, Canada).

For the purpose of the following invention, results are expressed in terms of wheat growth parameters, Se distribution and concentration in wheat plants and nutrient uptake effects, have been analysed to determine various effects of Se uptake by plants: a) Effects of Se on wheat growth parameters:

To check the effect of Se on plant growth several parameters were used: elongation of roots, weight of shoot and roots, and absorption of nutrient solution, which are connected with the needs of normal plant growth.

Results obtained when adding Se at different levels showed that enrichments up to 10 μΜ Se caused a slightly higher root elongation for selenate treatment (Fig. 1a). By contrast, selenite and mixture treatments at the same range showed no significant differences in root elongation in comparison with the control.

At higher Se concentrations root elongation decreased, reaching the minimum value at 100 μΜ Se, when visible toxic symptoms could be observed in shoots (strong chlorosis and wilting). These results showed an external Se effect concentration for a 50% inhibition of root elongation (EC50) of around 100 μΜ Se for selenite and selenate but not for the mixture of them.

As can be seen in Figure 1 b and 1c, the addition of Se in the range of 0-10 μΜ did not significantly affect root and shoot fresh weight. On the other hand, above 10 μΜ Se root and shoot fresh weight started to decrease significantly. This is in agreement with root elongation changes mentioned before.

In Figure 1d it can be seen an initial increase of volume of nutrient solution absorption followed by a decrease when Se concentrations applied were greater than 10 μΜ due to a poorer root absorption capacity at those concentrations. b) Se concentration and distribution in wheat plants

In Figure 2 are shown Se accumulation and distribution between roots and shoots. For the sake of clarity, these results have been divided into two different figures, related with total Se supplemented in the medium: Figure 2a, from 1 to 10 DM total Se concentration; and Figure 2b, from 50 to 100 DM total Se concentration. It is worthy to note that when selenate stimulates root elongation growth (1 and 10 μΜ treatments) Se accumulated in wheat plants is about 2.5-fold lower than that for selenite. However, when we worked with toxic selenate concentrations (50 and 100 μΜ treatments) Se contained in wheat exceeded 1.2-1.65 times those of selenite (Fig. 2).

The percentage of Se distribution kept constant with changes in Se concentration of each enrichment treatment. For selenite and mixture enrichments, Se is mainly accumulated in roots (80% and 70%, respectively), while for selenate the accumulation pattern was different depending on external concentrations. At the lowest selenate concentration (1 μΜ) Se was equally distributed between roots and shoots: with a 10 μΜ selenate supply Se levels were two fold higher in shoots than in roots and at the two highest external selenate concentrations, Se was mostly accumulated in shoots at the same percentages (85%). As it can be observed, the different pattern in total Se accumulation could be explained by a different rate of selenate uptake depending on its external concentration. The highest Se concentration found in roots was 335 g g-1 while in shoots was 580 g g-1 which corresponds to the individual highest external selenite and selenate concentrations, respectively. The shoot/root ratio of total Se content in wheat plants ranged from 0.74 to 5.98 for plants supplied with selenate and was less than 0.25 for plants supplied with selenite, while this ratio ranged from 0.31 to 0.42 when a mixture of Se forms was supplied. c) Nutrient uptake

The concentration of several important macro and micronutrient elements were analyzed for all treatments in order to evaluate the effect of different Se supplementation on nutrient uptake in the analyzed plants. The macronutrient concentration of Ca, K, Mg, P and S and the micronutrients Cu, Fe, Mn and Zn in roots and shoots of wheat plants for each treatment are shown in figures 3 and 4 respectively, excluding mixture treatments where no significant results were obtained. d) Effects on macronutrients

It is very important to notice that the increase or decrease in macronutrient concentration was never higher than 35% (Fig. 3), being Ca and S the most affected by Se supplementation. Results also show that, in any case, no redistribution of elements between root and shoot was observed. The loss of concentration in roots, when observed, was never compensated by the concentration in shoots.

As seen in Figure 3a, Ca root uptake was more affected by low Se exposures than under toxic levels (50 and 100 μΜ Se), especially when applied as selenate. In the case of shoots, minor changes in Ca concentration were observed.

In Figure 3b it can be observed that selenite had greater effect on K uptake than selenate at low exposures (up to 10 μΜ Se). On the other hand, at high Se levels (up to 50 μΜ) no significant differences were observed between Se forms. In shoots, K accumulation was not significantly altered by any Se addition.

In Figure 3c it is shown that root Mg uptake suffered different tendencies depending on Se form applied: selenite enrichment caused a decrease in Mg levels, reaching a plateau at 50 μΜ Se, while selenate decreased Mg uptake up to 10 μΜ Se but at higher exposures increased it till control Mg levels. Shoot Mg concentrations under Se exposure were similar to control levels except for 50 and 100 μΜ of selenite, where Mg levels were significantly lower. Both selenite and selenate caused a similar decrease in root P concentration at high Se supplementation (Fig. 3d). In shoots P level was increased by both Se forms at low exposures while was slightly decreased at high Se exposure, especially by selenate.

In Figure 3e it can be seen that S uptake by roots was not dependent on the Se species added but on Se concentration. At low Se supply (up to 10 μΜ) S uptake was slightly higher than in control, while for higher Se levels (50 and 100 μΜ), S content was lower than in control. In shoots all selenite treatments showed a similar behaviour as in roots, while selenate at high concentrations (50 and 100 μΜ) increased significantly shoot S accumulation. e) Effects on micronutrients

High selenate concentrations stimulated Cu and Mn root uptake by 12-24% and 30-35% respectively (Figs. 4a and 4c); whereas had no effect on shoot Cu accumulation but caused a 20% reduction on shoot Mn level. High selenite exposures (>10 μΜ) inhibited Cu and Mn accumulation in both roots and shoots with a great impact (ca. 50%) in shoot Mn level.

As can be seen in Figure 4b, the effect of Se addition on Fe uptake and accumulation was stronger in the form of selenate than of selenite. In roots 10 μΜ selenate causes a Fe reduction of about 50% that was partially recovered at higher concentrations almost reaching the control level. Shoot effects were less intense than those observed on roots.

The effect of Se in root Zn concentration was mainly noticed at Se levels above 50 DM, causing both species the same stunt, but behaviour in shoots was different: concentration of Zn was mainly unaffected by the presence of Se (Fig. 4d).

For the present invention, as can be seen in Table 2 (table fit results) the proportion of inorganic to amino acid selenium in wheat samples is dependent, not only, on the part of the plant analyzed, but also, on the form of the selenium initially supplied hydroponically. It is noteworthy that the low selenium concentrations in wheat are considered challenging for XAS analysis. That could explain that spectra from 1 μΜ selenium concentration treatments are noisier than those for the 10μΜ enrichments. Composition (%) Sum ( ) Residual

SeOj ^2" Se0 ₄ ^2" SeCy SeMet

1 μ Se0 ₃ ^2~ S 11 ,3 0.0 60,5 24,5 96.3 0,17

1 μΜ Se0 ₃ ^2" R 97 0.0 70,7 24,1 104,5 0,17

10μΜ Se0 ₃ ^2" S 16,8 0,0 55,8 35,4 108,0 0,16

10μΜ SeO^ R 6,9 0,0 50,8 42,3 100,0 0,07

1μΜ SeO S 0,0 32,5 5,7 61 ,9 100,1 0,49

1μΜ Se0 ^2' R 0.0 27,9 15,9 56,2 100,0 0,54

10μ SeO ^' S 0,5 50,9 0,0 48,6 100,0 0,21

10μ Se0 ₄ ^2' R 4,9 38,2 27,9 32,3 103,3 0,07

1 Μ mixture S 24,9 8,1 81 ,5 0,0 114,5 0,63

1μΜ mixture R 7,5 2,4 98,3 0,0 108,2 0,75

10μΜ mixture S 17,5 12,3 75,4 0,0 105,2 0,32

10μΜ mixture R 9,0 2,0 73,6 15,3 99,9 0,08

Table 2

Table shows linear combination fit results of the samples (R=root, S=shoot). Raw quantitative compositional results in percentage, sums of components for each sample and residual values corresponding to the quality of the fit.

Concerning selenite enrichment treatments, although it is observed the same selenium speciation distribution proportion in shoots and roots samples, the overall total selenium concentration is higher in the plant root as reported previously, this is in accordance with Mounicou et.al. results in the selenite enriched Brassica Juncea, a selenium enriched accumulator plant (Mounicou, S.; Vonderheide, A.P.; Shann, J.R.; Caruso, J.A. Comparing a selenium accumulator plant (Brassica juncea) to a nonaccumulator plant (Helianthus annuus) to investigate selenium-containing proteins. Anal. Bioanal. Chem. 2006, 386, 1367-1378). As expected in the selenite fortification, no selenate is present in any part of the plant, so it basically reduces selenite to organoselenium compounds as SeMet and SeCy, being the latest the major selenocompound in this case.

For selenate enrichment treatments a small percentage of selenium remains as selenite (1- 5%), i.e. a significant percentage of selenate is not metabolized (27-50%) but the metabolized amount is easily reduced to the corresponding organic selenium compounds as SeCy and SeMet. As for the selenite enrichments SeCy is the major selenoaminoacid, in the case of selenate enrichments it is the minor, being SeMet the predominant form.

In the case of mixed selenite and selenate treatments results show that the amount of inorganic selenium is higher in shoots compared with roots. On the other hand, looking at the organoselenium content, selenium is mainly accumulated in both roots and shoots as SeCy except in 10μΜ enriched roots where a small percentage of SeMet is present.

Concerning Se biofortification, it is noteworthy that we are looking for Se enrichment procedures that mainly accumulate organic selenium forms in shoots, so shoots are the first stage of wheat growth and they will determine the Se wheat content that is going to be used for the manufacture of Se enriched products. Comparing all selenium enrichment treatments, enriching with selenate we obtain the lower selenium organic content in shoots, on the other hand, the selenite one accumulates a higher organic selenium content in shoots as previously reported have shown toxicity symptoms when wheat is exposed to that treatment. In mixture treatments the selenium total organic content in shoots is higher than in selenate though slightly lower than in selenite, however, it was proved above that mixtures attenuate selenite toxicity. To conclude, results have shown that the best way to anthropogenically enrich selenium in order to obtain a higher organic selenium content in shoots and no toxicity symptoms is mixing both selenite and selenate species at equal concentrations (5μΜ Se as selenite and 5μΜ Se as selenate).

On the other hand, obtained results reveal that the variation of Selenite/Selenate ratio produces a significative effect on the nature of aminoacids obtained. Thus in Table 2, it is possible to observe how the production of Selenomethionine increases in favor of Selenocysteine when the Selenite/Selenate ratio decreases. This effect correlates the redox conditions of the hydroponic solution provided by the Selenite/Selenate ratio with the observed redox behavior of the corresponding selenoaminoacids Selenocysteine and Selenomethionine observed at the related XANES Spectra. This finding provides us a way to tune chemically the conditions for a specific selenoaminoacid production.

Therefore, for the present invention, these observed effects in Figure 1 can also be seen on the fresh weight of both roots and shoots and on the amount adsorbed from plant solution. In addition, these results confirm a duality of Se effects concerning both the level of concentration applied to the nutrient solution and the chemical form of Se used to feed plants. Such duality of Se concentration has been previously reported in other works (Hopper and Parker, 1999; Hartikainen et al. 2000; Cartes et al. 2005; Feng et al. 2009) but they have only tested individual Se forms (selenite or selenate) not their mixtures. The toxicity observed can be interpreted as the irruption of Selenium species on both the amino acids generation process (ref) and possibly on the cellular energy production path.

Surprisingly, the presence of both Se species mixed together in nutrient solution at highest concentrations tested (25+25 and 50+50) show lower toxicity than selenite alone at 50 or 100 μΜ, suggesting that selenate attenuates toxicity of selenite in mixtures.

On the other hand, most plants contain less than 25 g Se g- ¹ dry matter and are termed non-accumulators (White et al. 2004). Such plants absorb Se but are incapable of tolerating high Se in their tissues and environment, thus Se toxicity occurs below about 10-100 g Se g- ¹ dry matter, although the exact value depends critically upon the selenate:sulfate molar ratio in the rhizosphere solution. These plants tolerate low Se concentrations in the rhizosphere by restricting Se uptake and movement to shoots (Wu and Lag, 1988; Wu and Huang, 1992). Spring wheat is considered a non-Se-accumulator but it has relatively high tolerance when compared with other non-accumulator plants because Se toxicity occurs above 100 g Se g- ¹ dry matter. The ability of wheat plants to accumulate and translocate Se from roots to shoots is highly dependent on the chemical form of Se and on the amount of Se applied to the nutrient solution as it is already reported in other works (Li et al. 2007; Govasmark et al. 2008; Feng et al. 2009).

Concerning total Se concentration in whole plant, two different behaviours could be observed: at low external Se levels (1 and 10 μΜ) the highest total Se concentration was observed in selenite cultures, while at high external Se levels (50 and 100 DM) it was selenate exposed plants which exhibited a higher concentration. Surprisingly, at low external Se concentrations Se level in plants treated with mixtures was always a bit lower than total Se of plants treated with selenite but higher than the level found in plants treated with selenate. Even more, at high external Se supply Se level in plants exposed to mixtures was always lower than those of plants treated with individual Se forms. These results could be due to a possible interaction between selenite and selenate. It seems that selenite uptake is favoured in front of selenate because most Se accumulated from mixture treatments is found in roots, organ where selenite preferentially accumulates when individually applied. The intensity of the interaction remains constant with increasing external Se mixture concentration as can be seen by the constant Se distribution in all Se mixture treatments (25-30% shoots / 70-75% roots). Thus, low Se tissue level found in plants treated with high Se mixture concentrations is probably due to a greater translocation of Se at high external selenate levels but not to a major interaction between different Se species.

These differences in Se uptake and movement in different parts of plant due to the variation in the species of Se applied to the media suggests that the bioassimilation of each Se species follows its own metabolic pathway.

Also, the observed efficiency of wheat plants to transport selenate to shoots (80%) can be due to two complementary mechanisms: a way of protecting the plant against selenate toxicity by accumulating it in leave vacuoles and, at the same time, a way that facilitates part of its further elimination by formation of Se volatile species. Se compartmentalized in leave vacuoles is less toxic because it has poor mobilization and incorporation into proteins. In our experiments selenite tends to accumulate in roots more than in shoots which is in accordance with the literature (Hopper and Parker, 1999; Feng et al. 2009) and only about 20% of selenite taken up has been transported to shoots which is comparable to the small fraction found by Arvy (1993) and the 10% reported by De Souza et al. (2000) in dicotyledonous species. This unequal distribution can be explained by the non-enzymatic transformation of selenite to toxic organoselenium species, which are not transported from roots to shoots (De Souza et al. 2000). Organic Se forms can readily enter sulphur assimilation pathways, leading to more Se substitution into proteins (Smith and Watkinson, 1984; De Souza et al. 1998). At shoot Se comparable levels (10 μΜ), Se from selenate treated plants stimulated root growth while selenite did not. Similar results were observed in ⁷⁵ Se-selenate and ⁷⁵ Se-selenite treated excised roots of Astragalus lentiginosus, a non-accumulator (Shrift and Ulrich, 1969). When plants were treated with selenate, 98% of accumulated Se remained as selenate, whereas under selenite treatment, plants retained about 51 % selenite, 23% selenate and only 19% in neutral or basic form, postulated to be SeMet. These data suggest that some plants are able to oxidize selenite back to selenate in small amounts.

In the case of mixture experiments, distribution between roots and shoots resembles more to the one of selenite than selenate. This may suggest that the presence of selenite in the medium blocks the transportation of selenate to shoots. This observation, together with the fact that selenite accumulates mainly in roots, can be the explanation for the parallelism observed for plants supplemented with only selenite and mixtures in terms of growth parameters related with roots (root elongation and fresh weigh, and absorbed nutrient solution). In general, the transport of Se from roots to biomass is closely connected with the chemical form of applied Se as reported in (Zayed et al. 1998). Further studies will be necessary to elucidate, not only the distribution of Se species applied for the enrichment, but also Se speciation in each part of the plant to better understand the Se bio-assimilation process.

In connection with the effects of Se on nutrient uptake; in general trends the inventors can observe that the uptake of macronutrients was less affected by the presence of Se than that of micronutrients, as already expected if we take into account the important role of these macronutrients in plant surviving. Se effect on roots was different from its effect on shoots; and those elements which mainly accumulate in roots (i.e. Ca, Fe, Zn and Mn) were more sensible to Se presence.

Ca presence is essential in the cell wall and together with K are in charge of membrane integrity thus the increased Ca and Mg level with increasing high Se concentration (Figs. 3a and 3c) could be a way to re-establish the integrity of root membranes under severe Se toxicity.

The fact that shoot P level is more affected by selenate than by selenite which is the form that has chemical similarity with phosphate suggests that selenite is not interfering with P uptake at low concentrations but at high Se:P ratios an increase in competition together with other metabolic interactions are responsible of the decreased tissue P level.

Our results are in accordance with that from Wu and Huang (1992) with tall fescue, a grass like wheat, where Ca concentration was increased but P concentration was decreased under Se stress and with those from Hopper and Parker (1999) who found an inhibition of P uptake by Se in Lolium perenne and Trifolium fragiferrum.

The extraordinary S uptake and translocation to shoots at high selenate treatments (Fig. 3e) is in agreement with the findings of Bell et al. (1992). A comparative study conducted by these authors on the antagonism between selenate and sulfate concluded that Se accumulators preferentially absorb Se in the face of competition with sulfate but non-accumulators discriminate against selenate uptake relative to sulfate. They observed that the non-accumulator plant had increased shoot S concentration by increasing selenate in solution, but only when shoot Se was above 20 g g- ¹ DW. In our study wheat plants (non-accumulator) that presented these high shoot S level have above 250 g Se g- ¹ DW while plants with no change in shoot S levels have less than 30 g Se g- ¹ . Selenate-induced stimulation of S uptake may be a result of incipient Se toxicity, which is in accordance with root-elongation and growth-inhibition by this Se species.

It is proved that expression regulation of high-affinity sulfate transporter genes (HAST) is mediated by S plant status, where elevated concentrations of sulfate and glutathione (GSH) down-regulate transcription and O-acetyl-L-serine (OAS) increases transcription (Hirai et al. 2003; Maruyama-Nakashita et al. 2003). In our nutrient solution sulfate level could be suboptimum for wheat and with increasing selenate exposure competition between both ions increases causing induction in the expression of HAST transporters. White et al. (2004) suggested that inducible high affinity sulfate transporters are more selective for sulfate than constitutively expressed low affinity transporters in the non-accumulator A thaliana. Their work also provides further evidence that competition between Se and S for assimilation and incorporation into proteins contributes to Se toxicity symptoms in plants.

Phytotoxicity of high Se levels is associated mainly with non-specific replacement of sulphur amino acids by their Se analogues (Terry et al. 2000). Moreover, this effect may result from disturbances in mineral balance, which is indicated by the analysis of nutrient composition of wheat plants performed in the present experiment. Toler et al. (2007) found in Brassica oleracea a positive correlation between selenate concentration in solution and Se and S uptake.

Although Cu and Mn are essential trace elements for plant development and growth, they are toxic when in excess (Pittman 2005). Many heavy metals stimulate the formation of free radicals and render oxidative stress, either by direct electron transfer or inhibition of normal metabolic reactions (Hall 2002; Metwally et al. 2005).

Concerning micronutrients, their uptake was affected by Se presence in different ways. Fe uptake showed a differential behaviour probably due to its high concentration in plants compared with other micronutrients. In both roots and shoots, all selenate treatments caused a reduction of Fe uptake. Clorosis observed in plants exposed at toxic Se levels can be a consequence of the strong Se-interaction with Fe, Mn and Cu uptake. Molnarova and Fargasova (2009) found a strong photosynthetic pigment reduction in Triticum aestivum under Se exposure that can be related with low Fe levels in shoots.

Cartes et al. (2005) and previously Hartikainen et al. (2000) reported the duality of Se effects on the antioxidative system in ryegrass; at low concentrations Se acted as an antioxidant whereas at shoot Se concentrations above 20 mg kg- ¹ DW it acted as a pro-oxidant by increasing lipid peroxidation and reducing yields. In our study, wheat presented toxicity symptoms and significant reduced root elongation growth at shoot Se concentrations above 30 mg kg- ¹ DW while below this concentration it had a stimulant effect on root growth and biomass production which is in accordance with the previously discussed.

Results obtained allow to conclude that both Se species tested, selenite and selenate, behave differently when absorbed by wheat plants in hydroponics culture, and the effects in plants are strongly dependent on Se species concentration in solution. However, the uptake of macronutrients was not strongly affected by the presence of any Se form, while the effect in micronutrient uptake was directly related with the element studied, without observing a common behavior for all checked elements.

It is remarkably that the largest differences in nutrient content we determined, as compared to control plants, were closely connected with high tissue Se levels and a considerable decrease in dry matter of plants and a reduction of root growth. Thus, regarding to Se supplementation of wheat plants, the form of Se applied should be taken into account as well as the total Se concentration.

In connection with, aspects of Selenium uptake mechanism; the observed differences on the distribution of Se in wheat plants after the uptake of corresponding inorganic forms of Selenium, indicate that possible interconversion of selenate to selenite or viceversa is very improbable in both the hydroponic solution or in the wheat plant tissues. So, it looks like a different path for the absorption of each selenium species is taking place. Thus, transport of selenate is more active than selenite, since Se is mostly accumulated on the plant shoots when using selenate in the hydroponic solution, while using selenite, Se is accumulated in the plant roots, what, according to Xiao-Zhang Yu, Ji-Dong Gu (2008), reveals a possible dependency on plant transpiration. In addition, Hua-Fen Li, Steve P. McGrath and Fang-Jie Zhao (2008) reported that the uptake of selenite depends on the related metabolism process. Thus, they indicate that spite of the similarity between the uptake rates of selenite and selenate by wheat, selenite is faster assimilated into organic forms in roots than selenate, having a lower mobility in xylem transport than selenate that is not readily assimilated into organic forms and will circulate fast in xylem transport. These properties can explain our results on the different distribution of Se in wheat plants when feeding them with each one of the two different Se species. Results corresponding to the mixture of the two species also support the interpretation of the individual behaviour, so Se is mostly accumulated in roots because of the explained fast selenite uptake and also shoots are less loaded since selenite seems to hinder the transport of selenate in the xylem, as reported by the indicated authors.

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