Direct and indirect selenium speciation in biofortified wheat: A tale of two techniques

Abstract

Wheat can be biofortified with different inorganic selenium (Se) forms, selenite or selenate. The choice of Se source influences the physiological response of the plant and the Se metabolites produced. We looked at selenium uptake, distribution and metabolization in wheat exposed to selenite, selenate and a 1:1 molar mixture of both to determine the impact of each treatment on the Se speciation in roots, shoots, and grains. To achieve a comprehensive quantification of the Se species, the complementarity of high-performance liquid chromatography coupled with inductively coupled plasma mass spectrometry and X-ray absorption spectroscopy was exploited. This approach allowed the identification of the six main selenium species: selenomethionine, selenocysteine, selenocystine, selenite, selenate, and elemental selenium. The three treatments resulted in similar total selenium concentration in grains, 90-150 mg Se kg^-1 , but produced different effects in the plant. Selenite enhanced root accumulation (66% of selenium) and induced the maximum toxicity, whereas selenate favored shoot translocation (46%). With the 1:1 mixture, selenium was distributed along the plant generating lower toxicity. Although all conditions resulted in >92% of organic selenium in the grain, selenate produced mainly C-Se-C forms, such as selenomethionine, while selenite (alone or in the mixture) enhanced the production of C-Se-Se-C forms, such as selenocystine, modifying the selenoamino acid composition. These results provide a better understanding of the metabolization of selenium species which is key to minimize plant toxicity and any concomitant effect that may arise due to Se-biofortification.

FIGURE 1

(A) Roots, shoots, and grains dry weight for each Se treatment, and (B) number of spikes, number of grains per spike and average dry weight of a single grain for each Se treatment expressed as relative percentage respect to the control plants (represented as mean ± SE, n = 8). Letters indicate significance (p < 0.05) between different treatments.

FIGURE 2

Phytohormone concentration in mg kg⁻¹ DW in roots and shoots for jasmonic acid (JA), salicylic acid (SA), 3‐indoleacetic acid (IAA) and abscisic acid (ABA), represented as mean ± SD (n = 3). Letters indicate significance (p < 0.05) between different treatments.

FIGURE 3

Selenium concentration in roots, shoots, and grains (mg Se kg⁻¹ DW) represented as mean ± SD (n = 8). Letters indicate significance (p < 0.05) between treatments.

FIGURE 4

Sulfur and phosphorus concentration in roots, shoots, and grains (g kg⁻¹ DW) represented as mean ± SD (n = 8). Letters indicate significance (p < 0.05) between treatments.

FIGURE 5

Chromatograms of the standard selenium species: selenocystine (SeCyst), methylselenocysteine (MeSeCys), selenomethionine (SeMet), sodium selenite (Se(IV)), and sodium selenate (Se(VI)) (top), and chromatograms of roots, shoots and grains of wheat samples enriched with selenite, selenate and mixture of both species (bottom), using the optimized gradient method of Table S1. The chromatograms have been shifted vertically for the sake of comparison.

FIGURE 6

Normalized Se K‐edge XANES spectra of roots, shoots and grains enriched with selenite, selenate and mixture treatments grouped by tissue type and XANES spectra of the reference compounds: Elemental selenium (Se(0)), selenocystine (SeCyst), methylselenocysteine (MeSeCys), selenomethionine (SeMet), sodium selenite (Se(IV)) and sodium selenate (Se(VI)). The spectra have been shifted vertically for the sake of comparison.