Received: 11 August 2021 Revised: 26 November 2022 Accepted: 12 December 2022 DOI: 10.1111/ppl.13843 ORIGINAL RESEARCH Physiologia Plantarum Direct and indirect selenium speciation in biofortified wheat: A tale of two techniques Maria Angels Subirana 1 | Roberto Boada 1 | Tingting Xiao 1 | Mercè Llugany 2 | 1 Manuel Valiente 1 GTS-UAB Research Group, Department of Chemistry, Faculty of Science, Universitat Abstract Autònoma de Barcelona, Bellaterra, Spain Wheat can be biofortified with different inorganic selenium (Se) forms, selenite or 2 Plant Physiology Group (BABVE), Facultat de Biociències, Universitat Autònoma de selenate. The choice of Se source influences the physiological response of the plant Barcelona, Bellaterra, Spain and the Se metabolites produced. We looked at selenium uptake, distribution and Correspondence metabolization in wheat exposed to selenite, selenate and a 1:1 molar mixture of Mercè Llugany, Plant Physiology Group both to determine the impact of each treatment on the Se speciation in roots, shoots, (BABVE), Facultat de Biociències, Universitat Autònoma de Barcelona, 08193 Bellaterra, and grains. To achieve a comprehensive quantification of the Se species, the comple- Spain. mentarity of high-performance liquid chromatography coupled with inductively Email: merce.llugany@uab.cat coupled plasma mass spectrometry and X-ray absorption spectroscopy was exploited. Funding information This approach allowed the identification of the six main selenium species: seleno- China Scholarship Council, Grant/Award Number: 201608330235; European methionine, selenocysteine, selenocystine, selenite, selenate, and elemental selenium. Synchrotron Radiation Facility (at BM25A The three treatments resulted in similar total selenium concentration in grains, SpLine), Grant/Award Number: 25-01-967; 1 H2020 Marie Skłodowska-Curie Actions 90–150 mg Se kg , but produced different effects in the plant. Selenite enhanced (European Union), Grant/Award Number: root accumulation (66% of selenium) and induced the maximum toxicity, whereas 665919; Ministerio de Economía y Competitividad (MINECO, Spain), selenate favored shoot translocation (46%). With the 1:1 mixture, selenium was dis- Grant/Award Numbers: BES-2013-066639, tributed along the plant generating lower toxicity. Although all conditions resulted in CTM2015-65414-C2-1-R(CHEMNEXUS) >92% of organic selenium in the grain, selenate produced mainly C-Se-C forms, such Edited by J.K. Schjoerring as selenomethionine, while selenite (alone or in the mixture) enhanced the produc- tion 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. 1 | I N T RO DU CT I O N conditions (e.g., heart disease, type-2 diabetes, neuromuscular disor- ders, depression) (Rayman, 2000); it is involved in the functioning of Selenium (Se) is an essential micronutrient for humans since it is found the immune system and the production of the active thyroid hormone as selenocysteine in 25 human selenoproteins (D'Amato et al., 2020). (Rayman, 2000), it is effective in the chemoprevention of specific can- This amino acid is located in the active sites of enzymes and performs cers (Weekley & Harris, 2013), and it may inhibit viral expression and catalytic redox reactions, supporting various physiological functions virulence of HIV, influenza and COVID-19 (Gong et al., 2020; (Rayman, 2000). Although Se has a predominant role in antioxidant Rayman, 2012; Zhang et al., 2020). Hence, an appropriate Se dietary activity, it also contributes to mitigate several pathophysiological intake can highly benefit human health. This is an open access article under the terms of the Creative Commons Attribution-NonCommercial License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes. © 2022 The Authors. Physiologia Plantarum published by John Wiley & Sons Ltd on behalf of Scandinavian Plant Physiology Society. Physiologia Plantarum. 2023;175:e13843. wileyonlinelibrary.com/journal/ppl 1 of 14 https://doi.org/10.1111/ppl.13843 2 of 14 SUBIRANA ET AL. Physiologia Plantarum The Se content of food determines the Se status of the popula- benefits (Rayman et al., 2008). In general, inorganic Se species, such tion, but the range of adequate consumption is narrow, and both as selenite (Se(IV)) and selenate (Se(VI)) ions, are more toxic than their excessive and deficient ingestion may lead to Se-related diseases and organic counterparts (Rayman et al., 2008). In addition, they have low medical conditions. Regular consumption of food containing between bioaccessibility (e.g., Se(IV)) and low bioavailability (e.g., Se(VI)) 0.1 and 1 μg Se g 1 is considered adequate to reach the recom- (Fairweather-Tait et al., 2010). Oppositely, organic Se is more bioavail- mended 55 μg Se day 1 (Dumont et al., 2006). Unfortunately, cases able and bioactive, as more than 90% of Se consumed in the form of of moderate deficiency are rather common, and it has been estimated selenoamino acids is absorbed by the body, resulting in an effective that around one billion people may be Se deficient (Hawkesford & increase of blood Se levels and GPx enzymatic activity (Fairweather- Zhao, 2007), with insufficient Se levels in their blood to optimize the Tait et al., 2010). Inorganic Se species are the predominant form of Se glutathione peroxidase (GPx) enzymatic activity (Rayman, 2000). This found in the environment. Selenate is more soluble and less absorbed can be attributed to low Se concentration in soils, which results in a and, consequently, more bioavailable for plant uptake (Yu et al., 2019), Se shortage through the food chain. Crop enrichment with whereas selenite is immobilized by adsorption on iron and aluminum Se-containing fertilizers has been proposed as the best solution for oxides and soil organic matter (Li et al., 2017). Thus, selenate is the Se-biofortification (Hawkesford & Zhao, 2007). Indeed, this strategy main species present in agricultural soils (Raina et al., 2021), and it is has already been successfully applied in Finland, where the Se level in usually the choice for agronomic fortification through Se-enriched fer- blood serum of the population noticeably improved (Alfthan tilizers over selenite and the much more expensive organic forms et al., 2015), as well as in the USA, the UK, Australia and China (Mora (Alfthan et al., 2015). et al., 2015). Crop plants can take up selenite and selenate species through Although Se is not essential for land plants since it does not fulfill active membrane transporters in roots and leaves, but the two inor- a specific role in their metabolism (Hawkesford & Zhao, 2007), plants ganic ions have distinct behavior. They follow different transport can tolerate and even thrive on limited amounts of Se. Above a certain pathways (Raina et al., 2021; Zayed et al., 1998), which may influence threshold, which depends on the plant accumulation capacity, toxic the distribution and accumulation through the plant organs and the Se effects may appear, which can depress plant growth (Guerrero species formed. Plants can metabolize these inorganic ions into et al., 2014). Despite the efforts that have been devoted to Se biofor- organic Se species, such as selenomethionine (SeMet), selenocysteine tification studies, several factors, such as the focus on hyperaccumula- (SeCys), selenocystine (SeCyst), methylselenocysteine (MeSeCys), tor plants, the use of high Se doses and the lack of a consistent gamma-glutamyl-methylselenocysteine, dimethylselenide, dimethyldi- strategy in the selection of Se source, have made it difficult to reach selenide and selenocystathionine (Freeman et al., 2006; Winkel of a consensus towards a widespread methodology for Se biofortifica- et al., 2015; Xiao et al., 2020). tion (D'Amato et al., 2020). The main Se species in wheat are the amino acids SeMet, Cereals, which are considered non-accumulators, have an average MeSeCys and SeCyst, together with unmetabolized inorganic 1 Se content of 0.01–0.55 mg kg fresh weight (Hawkesford & Se(IV) and Se(VI) (Wang, Ali, et al., 2020). Elemental selenium (Se(0)) Zhao, 2007). Nevertheless, Se concentrations in wheat grown in sele- has also been reported to be present in wheat (Xiao et al., 2021). 1 niferous soils can be as high as 30 mg kg dry weight (DW) (Lyons Although the literature regarding wheat biofortification with Se is 1 et al., 2005; Whanger, 2002), and exceptionally up to 62 mg kg extensive, most research has only focused on the total Se content of DW, in South Dakota, USA (Cubadda et al., 2010), and up to wheat grain (Broadley et al., 2010; Zou et al., 2019) and addressing 1 387 mg kg DW in Punjab, India (Eiche et al., 2015). Indeed, wheat the importance of Se speciation in wheat grain has only achieved par- has the highest Se accumulation capacity among cereals (Raina tial results. SeMet is reported to be the most abundant species in et al., 2021) and can store considerably high amounts of Se when wheat grain, with values generally over 70% of the total Se for both enriched without a significant decrease in yield due to toxicity (Wang, native and supplemented grains, regardless of the amount, chemical Ali, et al., 2020). Since wheat is the second most important food crop form and application method of the Se biofortification (Cubadda and is already a major dietary source of Se (Boldrin et al., 2016), wheat et al., 2010; Galinha et al., 2015). Selenate is also commonly quanti- is a good candidate for producing Se-biofortified food. Furthermore, fied, with literature reporting values below 5% (Galinha et al., 2015; wheat is one of the most produced and consumed cereals worldwide Warburton & Goenaga-Infante, 2007). An extremely limited number (Enghiad et al., 2017), and it is already one of the main sources of of studies have achieved a successful determination of the five Se nutrients in diets (Hussain et al., 2010). In addition, the Se- species (Di et al., 2023; Eiche et al., 2015; Hart et al., 2011; Wang biofortification of wheat has already been proven to be retained in et al., 2022; Wang, Ali, et al., 2020; Xiao et al., 2021), and the present the production of enriched wheat-based foods such as flour and bread study is the first to quantify six species in wheat. Likewise, very few (Hart et al., 2011), pasta (Poblaciones et al., 2014), and soup (Bañuelos have used complementary techniques to confirm species identifica- et al., 2022). tion and validate the applied method (Aureli et al., 2012; Warburton & Regarding the metabolization of Se in humans, since different Goenaga-Infante, 2007). The majority of the speciation studies have chemical species have different bioavailability and toxicity, their exclusively employed high-performance liquid chromatography with chemical form, rather than the amount consumed, determines the inductively coupled plasma mass spectrometry (HPLC-ICP-MS), which, final Se status and, ultimately, the associated nutritional and health in some cases, resulted in an incomplete characterization of the SUBIRANA ET AL. 3 of 14 Physiologia Plantarum overall species due to the low stability of certain Se species after the spectrometry (ICP-MS) and HPLC-ICP-MS, respectively. Two cultures required sample pre-treatment steps and incomplete recoveries of the were performed in two different years under the same conditions. methodologies used (Połatajko et al., 2005; Whanger, 2002). These The results were averaged since the two cultures did not present sig- drawbacks can be overcome by direct speciation techniques such as nificant differences resulting in eight plants per treatment. X-ray absorption spectroscopy (XAS) which allows Se speciation in After harvesting, roots were washed with ice-cold CaCl2 solution solid form without requiring extraction and pre-treatment steps. In to remove the elements from the root apoplast and then rinsed with general, the use of direct speciation techniques and/or combined deionized water. The plants were divided into roots, shoots (including approaches for Se analysis in plants is scarce (el Mehdawi et al., 2014; stems and leaves) and spikes, weighted and stored at -20 C until fur- Freeman et al., 2006; Pickering et al., 2000), and only a few have stud- ther processing. ied wheat plants (Wang et al., 2015; Xiao et al., 2020, 2021). Thus, a multidisciplinary approach is necessary for unraveling the complete chemical state of Se in plants to comprehend the Se metab- 2.2 | Plant physiological growth parameters olism and ultimately address how Se accumulation in plants should be optimized to improve the specific nutritional value of Se-enriched Plant material was oven dried at 45 C for 4 days until reaching stable food (Raina et al., 2021). In this context, the objective of the present weight. The physiological effects of the different Se forms on wheat work is to use the complementary speciation information obtained plants were evaluated by the biomass produced in terms of the per- from both HPLC-ICP-MS and XAS techniques to quantify six selenium centage of the dry weight of roots, shoots (stem and leaves) and species: SeMet, MeSeCys and SeCyst, Se(IV), Se(VI) and Se(0), to grains of the treated plants compared with control ones. Moreover, understand the different Se pathways in wheat depending on the the effect on grain yield was tracked as the number of spikes per inorganic Se source supplied. This has been accomplished by studying plant, grains per spike and grain weight. the distribution of the Se species along the plant (roots, shoots, and The accumulation of Se in roots, shoots and grain was determined grains) to completely characterize the output of the Se biofortification as well as the Se translocation factor, which is calculated as the shoot process, as well as the effects of this biofortification on the plant Se concentration divided by the root Se concentration. physiological growth, nutrient concentration, and hormonal homeo- The significance of the results was assessed by an ANOVA statis- stasis. This information is essential to develop the best strategy to tical analysis with 95% confidence with the TIBCO Statistica software improve the crop yield and the production of Se-enriched wheat- (StatSoft). based functional food to tackle human Se deficiency. 2.3 | Hormone analysis 2 | MATERIALS AND METHODS The plant hormones (±)-jasmonic acid (JA), salicylic acid (SA), 2.1 | Wheat culture (+)-cis, trans-abscisic acid (ABA) and 3-Indoleacetic Acid (IAA) were analyzed by LC-ESI-MS/MS in multiple reaction monitoring n purchased Plants of common wheat (Triticum aestivum L. cv. Pinzo mode (MRM). These phytohormones were extracted following the  S.A.) were hydroponically grown. Seeds were ger- from Semillas Fito methods of Llugany et al. (2013) and Xiao et al. (2020). Briefly, minated in moistened filter paper at room temperature for 5 days. 250 mg of frozen roots and shoots were ground in an ice-cold mor- Then, seedlings were transferred to opaque plastic containers filled tar and repeatedly extracted with 750 μl Methanol:2-Propanol: with continuously aerated, modified ½ strength Hoagland's nutrient Acetic acid (20:79:1, v/v/v). The supernatant was recovered after solution buffered with MES (2-(N-morpholino)ethanesulfonic acid, centrifugation and lyophilized. Then, the obtained pellets were dis- C6H13NO4S) to maintain a stable pH of 6.0 (Guerrero et al., 2014). solved in 250 μl of methanol and filtered with a Spin-X centrifuge The culture was carried out in a controlled-environment growth tube filter of 0.22 μm cellulose acetate (Costar, Corning chamber with 18-24 C temperature, 60–70% humidity and 320 μE Incorporated,). 2 1 m s light intensity with a short photoperiod of 8 h light/16 h Quantification was done using a standard addition calibration darkness during vegetative growth and a long photoperiod of curve spiking control plant samples with standard solutions ranging 12–16 h light/8–12 h darkness for flowering induction and grain from 50 to 1000 ppb for SA and 5 to 100 ppb for JA, ABA, and IAA. production. Deuterated hormones (±)-jasmonic acid-d6 (JA-d6), (+)-cis, trans- Two weeks old plants (four per treatment), with three leaves abscisic acid-d6 (ABA-d6) and Indole d5-acetic acid (IAA-d5) at 30 ppb unfolded, were exposed to sodium selenite, sodium selenate or a 1:1 and salicylic acid-d6 (SA-d6) at 300 ppb were used as internal stan- mixture of both at 10 μM. A control treatment without Se was also dards in all the samples and standards measurements. All standards included. The solution was renewed weekly over 12 weeks until were purchased from (Sigma-Aldrich). Separation was done using an senescence to maintain constant water, nutrients, and Se levels. The HPLC Agilent 1100 (Waldrom) on an Acquity UPLC BEH C18 elemental concentrations and stability of the species in the nutrient 2.1  100 mm ID, 1.7 μm column (Waters, USA) at 50 C with a con- solution were monitored by inductively coupled plasma-mass stant flow rate of 0.8 ml min 1 and 10 μl injection. The elution was 4 of 14 SUBIRANA ET AL. Physiologia Plantarum performed with a gradient between 0.1% of formic acid in methanol 2.6 | Direct selenium speciation and 0.1% of formic acid in Milli-Q water, as detailed in Xiao et al. (2020). Detection was carried out with an API 3000 triple quadrupole X-ray absorption spectroscopy (XAS) provides element-selective mass spectrometer (Perkin-Elmer Sciex, Concord) using the Turbo Ion- chemical speciation information without the need for any sample pre- spray source in negative ion mode. treatment, thus avoiding issues due to incomplete recoveries and/or reactivity of the species. XAS measurements at Se K-edge were per- formed at the BM25A SpLine beamline of the European Synchrotron 2.4 | Total selenium and mineral nutrient analysis Radiation Facility (ESRF) using a Si(111) double-crystal monochroma- tor. Energy calibration was done with Se(0) to 12,658 eV (Ravel Ground dry plant material (50 mg) was acid digested in HP500 PFA et al., 2005). For the measurements, ground and dried material from  vessels with 4 ml of HNO3:H2O2 (3:1) at 180 C and 1.9 atm for roots, shoots or grains were pressed into pellets. The experiment was 45 min in a microwave digestion system (Mars 5, CEM). carried out at liquid nitrogen temperature to minimize radiation dam- 78 24 31 55 56 64 65 Determination of Se, Mg, P, Mn, Fe, Zn, Cu, and age. The fluorescence signal was collected by a 13-element 98 Mo concentrations was done by ICP-MS (X Series 2, Thermo Fisher Si(Li) solid-state detector (e2V Scientific Instruments). Reference sam- 45 69 89 115 Scientific) using Sc, Ga, Y, and In as internal standards. ples were prepared from pure Se standards of SeMet, SeCyst, The macronutrients S, Ca and K were measured by Induced MeSeCys, Se(IV), Se(VI), and Se(0) and measured in transmission mode Coupled Plasma Atomic Emission Spectroscopy at the “Scientific and using gas ionization chambers. Data reduction and normalization and Technological Centers of the University of Barcelona (CCiTUB)”. subsequent linear combination fitting (LCF) analysis were performed A certificate reference material (CRM), SELM-1 (NRC, Canada), with the Athena program of the Demeter software package (Ravel consisting of a Se-enriched yeast in which Se is found as SeMet, was et al., 2005). used to validate the sample digestion procedure and Se determina- 1 tion. The determined Se concentration of 1993 ± 39 mg kg (mean ± SD with n = 8) agreed with the certified value of 2059 3 | RE SU LT S 1 ± 64 mg kg (Mester et al., 2006). 3.1 | Selenium effect on plant development 2.5 | Conventional selenium speciation The physiological effects of Se exposure on wheat plants are shown in Figure 1 and Table 1. There is a significant reduction in plant bio- Selenium species were determined by HPLC-ICP-MS (78Se elution). mass (43% shoots, 55% grain) in selenite-treated plants compared Previously, 50 mg of ground and dried plant material was extracted by control. Selenate and mixture treatments did not result in any statisti- enzymatic digestion with 10 mg of protease XIV and 5 ml of degassed cally significant change. Selenite treatment did not alter the number NH4H2PO4 (saturated with nitrogen to ensure oxygen depletion). The of stems, but it reduced the number of stems that were able to gener- flask was sealed and placed in an incubator for 16 h at 37 C, in dark- ate a spike compared with the control plants. Additionally, the number ness and with continuous stirring. Afterwards, samples were cooled of grains produced in each spike was not significantly affected,  down with an ice bath, filtered, maintained at 4 C, and analyzed although the average weight of each single grain is significantly lower within 4 h. with respect to the control or the other treatments (Figure 1B). Con- A PRP-X100 (Hamilton) strong anion exchange column, sequently, 10 μM selenite does not reduce the kernel formation in the 250  4.1 mm, with a stationary phase of 10 μm particle diameter, spike but impairs the kernel development and ripening to achieve a  was used for analysis at 20 C of 100 μl of sample, injected with an higher weight. On the other hand, selenate and mixture treatments HPLC Spectra system (Thermo Fisher Scientific). The mobile phase did not affect wheat yield. was ammonium citrate with 2% MeOH at pH 5.0. A gradient elu- tion was performed from 5 to 15 mM of ammonium citrate, as reported in Table S1. Peak identification and quantification were 3.2 | Selenium effect on phytohormones performed by external calibration and confirmed by spiking with commercial standards of sodium selenite, sodium selenate, seleno- Plant hormones JA, SA, IAA, and ABA are known to be implicated in methionine, methylselenocysteine hydrochloride and selenocys- signaling pathways that respond to abiotic stresses (Wang, Song, 115 tine and using In as an internal standard. A standard for et al., 2020). They were analyzed, and the results are shown in selenomethionine oxide was prepared by SeMet oxidation with Figure 2. The JA concentration was not significantly affected in roots 30 μl of H2O2, and a standard for selenomethionine selenone with or shoots by the addition of any form of selenium compared with con- 1000 μl of H2O2. trol, but shoots treated with selenite or with the mixture treatment Additionally, the SELM-1 CRM was used for the assessment of had significantly higher JA than the selenate treated plants. Salicylic the method as well as the extraction efficiency and chromatographic acid tended to decrease after selenite and selenate treatments, with a recovery. significant reduction in the roots of plants treated with selenate as SUBIRANA ET AL. 5 of 14 Physiologia Plantarum F I G U R E 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. T A B L E 1 Roots, shoots, and grains Control Selenite Selenate Mixture dry weight (g DW), number of spikes, number of grains per spike, and average Roots DW 1.5 ± 0.3 (a) 0.9 ± 0.2 (a) 1.4 ± 0.3 (a) 1.3 ± 0.2 (a) dry weight of a single grain (mg DW) for Shoots DW 22 ± 2 (a) 13 ± 1 (b) 20 ± 3 (ab) 19 ± 2 (ab) each Se treatment Grains DW 6.1 ± 0.6 (a) 2.7 ± 0.5 (b) 5.2 ± 0.7 (a) 5.1 ± 0.8 (a) No. of spikes 9.0 ± 1 (a) 6.1 ± 0.7 (b) 7.3 ± 0.6 (ab) 6.6 ± 0.6 (ab) No. of grains per spike 23 ± 3 (a) 19 ± 3 (a) 25 ± 2 (a) 24 ± 3 (a) Single grain DW 30 ± 1 (a) 20 ± 3 (b) 28 ± 0.8 (a) 32 ± 0.9 (a) Note: Results are shown as mean ± SE with n = 8. Letters indicate significance (p < 0.05) between different treatments. well as in the shoots of plants treated with selenite. The ± 0.04) treatments and higher than 1 under selenate exposure (2.57 3-indoleacetic acid level tended to decrease with selenium exposure, ± 0.35). Therefore, the translocation to shoots was favored over root and it was significantly reduced in the roots of both selenite and sele- accumulation only on selenate treatments. nate plants, as well as the shoots of selenite and mixture treatments. The Se concentration in roots was statistically different among Abscisic acid was not significantly affected by any of the treatments. the three treatments, being the highest in selenite biofortification. Shoots of plants treated with selenate had a significantly higher level of Se than shoots from selenite and mixture treatments. However, 3.3 | Selenium uptake and distribution despite the difference in the translocation from root to shoot between selenite and selenate, the final Se concentration in grain was The Se concentration in the roots, shoots, and grain is reported in not statistically different between the two treatments. Only the mix- Figure 3. The Se concentration in treated plants greatly differs from ture treatment resulted in a significantly lower selenium amount in 1 control plants, which demonstrates the successful uptake and accu- grains (95 ± 11 mg Se kg DW), in comparison with selenite (149 1 1 mulation of Se by wheat. The results reveal that the distribution of Se ± 29 mg Se kg DW) and selenate (145 ± 8 mg Se kg DW), which is not homogeneous through the different plant parts (roots, shoots, indicates that the simultaneous exposure to the two species does not and grain) and depends on the form of Se present in the substrate. In result in an additive effect. selenite-treated plants, Se was highly accumulated in roots (66% of the total Se), and very little was translocated to shoots (8% in stems and leaves, 26% in grains). In contrast, in selenate treatments, only 3.4 | Mineral nutrient analysis 18% of Se was accumulated in roots, whereas 46% was translocated to shoots and 35% to grains. Coherently, the mixture enrichment had The effects of Se over the assimilation of mineral macronutrients (S, P, an intermediate behavior, approximately 61% of the Se was found in K, Ca, Mg) and micronutrients (Fe, Mn, Cu, Mo, Zn) by wheat was roots, 15% in shoots, and 23% reached the grain. The translocation studied through their quantification in roots, shoots, and grain, and factor was lower than 1 for selenite (0.11 ± 0.01) and mixture (0.26 are represented in Figure 4 (for S and P) and Figure S1 (for K, Ca, Mg, 6 of 14 SUBIRANA ET AL. Physiologia Plantarum F I G U R E 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. lowered in the roots, but significantly increased in the shoots. The phosphorus concentration was significantly reduced in the roots of all selenium treatments, while no significant effects were seen in shoots and grains compared with control plants. In addition, selenite treatment resulted in higher shoot and grain K concentration and a lower root Ca amount than the control treat- ment. Selenite exposure did not modify the levels of Mg in wheat tis- sues. Selenate treatment did not significantly change K concentration but resulted in Ca levels that were lower in roots and higher in shoots compared with the control, whereas Mg levels were only higher in roots. The mixture treatment resulted in the same trends for K and Ca as observed for the selenate treatment, with no significant effects in K and lower concentration of Ca in root and higher in shoots. Mg con- centration was not significantly modified compared with the control. No significant effects were seen in Fe, Mn, Zn, Cu, and Mo con- centrations for the selenium-treated plants relative to the control. F I G U R E 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. 3.5 | Indirect speciation Fe, Mn, Cu, Mo, Zn). Selenite and selenate are known to be taken up The metabolization of the Se species was characterized by tandem and transported via sulfur and phosphorus transporters, and conse- HPLC-ICP-MS after enzymatic digestion to achieve a deeper under- quentially, those elements were the most affected by the selenium standing of the transformation of Se in wheat. As shown in Figure 5, exposure. Particularly, the sulfur concentration was mainly altered the five major species present in wheat tissues were identified to be with the selenate treatment, which caused S levels to be significantly SeMet, MeSeCys, SeCyst, Se(IV), and Se(VI). Their relative abundance SUBIRANA ET AL. 7 of 14 Physiologia Plantarum F I G U R E 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. 115 ± 8% grain) but not among the enrichment treatments (Figure S2). The employed methodology also achieved an extraction efficiency of 71.3 ± 0.9% from SELM-1 CRM. These values lie within the typical 70%–90% range for both SeMet and enzymatic digestions in general (Aborode et al., 2015; Maher et al., 2012). Shoots and grains show excellent extraction efficiencies, and con- sequently, Se is mainly found in the form of species that are readily soluble and extractable by mild extractions or by peptide bond cleav- age. On the contrary, the roots show very poor extractions, 50%, which could be explained by incomplete digestions due to the pres- ence of Se in forms not completely digestible with the enzymatic pro- tocol or insoluble, such as Se(0) (Aborode et al., 2015; Połatajko et al., 2005). On the other hand, the average chromatographic recovery for all tissues and treatments was 54 ± 20%, while the recovery for SELM-1 F I G U R E 5 Chromatograms of the standard selenium species: was 73 ± 3% (in agreement with the values reported for both plants selenocystine (SeCyst), methylselenocysteine (MeSeCys), and yeast [Połatajko et al., 2005; Maher et al., 2012]). The low selenomethionine (SeMet), sodium selenite (Se(IV)), and sodium recovery in plant tissues might be due to poor solubilization of sele- selenate (Se(VI)) (top), and chromatograms of roots, shoots and grains nopeptides and non-peptidic high molecular weight compounds or of wheat samples enriched with selenite, selenate and mixture of both species (bottom), using the optimized gradient method of Table S1. to non-specific interactions of Se species, resulting in forms that do The chromatograms have been shifted vertically for the sake of not give a well-defined peak or elute as a continuum during the comparison. chromatography (Połatajko et al., 2005). Consequently, due to lim- ited extraction efficiency for roots and incomplete chromatographic recoveries, only a fraction of the Se species in wheat tissues is quan- was dependent on the organ analyzed and the growing conditions. tified by HPLC-ICP-MS, but this technique provides a preliminary Additionally, a few minor unknown peaks were also detected, which insight into the selenium metabolism that occurs in wheat. represent 2%–15% of the total area. Regarding roots, chromatographic results from the extract always Selenoamino acid quantification by HPLC-ICP-MS requires care- showed SeMet as the most abundant species, as shown in Table 2 ful attention since sample pre-treatment may promote the oxidation and Figure 5. Selenite-enriched roots contained SeMet as 64 ± 5% of of SeMet to selenomethionine selenoxide. This yields a peak in the the total Se. The remaining inorganic Se was below the LOQ, but still, chromatogram close to the void-volume and to the SeCyst peak, it was detected that a small part of the Se was oxidized to selenate, which hampers an accurate determination (Cubadda et al., 2010; indicating a relatively oxidant environment in wheat roots. Oppositely, Galinha et al., 2015). Thorough care during the enzymatic digestion selenate-enriched roots had only 43 ± 7% of SeMet, with 27 ± 6% of has been shown to minimize the SeMet oxidation below 4 ± 2% in the Se remaining as selenate. However, very little selenite was detected. pure SeMet sample SELM-1 CRM. The extraction efficiency varied Consistently, in roots enriched by a Se mixture, selenite had been among the different tissues (50 ± 11% roots, 118 ± 34% shoots, and completely metabolized, but 10 ± 3% of selenate remained in the 8 of 14 SUBIRANA ET AL. Physiologia Plantarum 1 T A B L E 2 Selenium speciation Concentration (mg Se kg DW) determined by HPLC-ICP-MS in wheat SeCyst MeSeCys Se (IV) SeMet Se (VI) roots, shoots, and grains enriched with selenite, selenate, and mixture of the Roots Selenite > selenoamino acids > selenite (Gupta & Gupta, 2017; Zayed thermore, in non-enriched naturally seleniferous soils from Punjab, et al., 1998), which is in accordance with the results of this study. India, wheat cultivation resulted in Se concentrations up to 146 mg Se 1 1 In addition to the different transport and translocation pathways kg DW in vegetative tissues and 185 mg Se kg DW in grains for selenite and selenate, different genes and different loci have been (Cubadda et al., 2010), and, a subsequent investigation reported 1 1 shown to be involved in the tolerance mechanisms to each inorganic 196 mg Se kg DW in roots, 191 mg Se kg DW in stems and 1 Se specie in Arabidopsis plants (Van Hoewyk et al., 2008; Zhang 387 mg Se kg DW in leaves (Eiche et al., 2015). et al., 2006), also indicating a possible genetic factor in the distinct Regarding the chemical speciation shown in this work, the use of response of wheat to selenite and selenate. direct and indirect speciation techniques allowed the possibility of Furthermore, the metabolism of Se species is also uneven. Sele- obtaining complementary results. This was essential to validate the nate must be reduced to selenite before converting to selenide and speciation analysis performed by each method and, more importantly, subsequently to organic species (Hawkesford & Zhao, 2007; Winkel to quantify six different species in wheat, which cannot be done by a et al., 2015). In roots, the notable amount of unmetabolized selenate single technique. The quantification achieved with HPLC-ICP-MS is indicates that the reduction of selenate to selenite is a slow process, only partial since it only accounts for small soluble compounds, and but the absence of selenite indicates that, once selenite is formed, its thus, elemental selenium cannot be determined. Otherwise, XAS does reduction to organic selenium occurs rapidly. These facts confirm that not have this limitation, and all the species could be identified, but the the selenate to selenite transformation is the rate-limiting step of Se similar local atomic structure around Se of SeMet and MeSeCys makes metabolism in wheat (Terry et al., 2000; Wang et al., 2015; Zayed their distinction difficult. In this sense, XAS allowed us to confirm the et al., 1998). In other words, selenite has a shorter chemical path to presence of Se(0) in the roots treated with selenite and the mixture. Ele- the final selenoamino acid moiety than selenate. mental Se is present in wheat (Xiao et al., 2021) and the roots of other Coherently, selenate translocation to aerial parts occurs faster plants (Aborode et al., 2015; Valdez Barillas et al., 2012). Elemental Se is than its reduction, so selenate preferentially accumulates in shoots. A a plausible intermediate species when inorganic Se is reduced to sele- similar process occurs with sulfur. Sulfate is assimilated and reduced nide before further metabolization to organic Se (Winkel et al., 2015). in chloroplasts, but when the concentration in the xylem is too high, it Additionally, certain enzymes can also generate Se(0) and alanine from is also stored in the vacuoles of the leaf mesophyll cells (Hopper & SeCys (Ellis & Salt, 2003), and microorganisms could also play a role in Parker, 1999). Sulfate in the vacuoles is not metabolized, is innocuous its production. Since Se(0) is biologically inactive (Hopper & to the plant, and is rarely remobilized (Hopper & Parker, 1999). The Parker, 1999), the formation of this insoluble form when Se is supplied same phenomenon occurs with selenate, which accumulates as selenite could be a mechanism to counteract the stress from the 12 of 14 SUBIRANA ET AL. Physiologia Plantarum excessive accumulation in roots. Concerning grains, all treatments manuscript writing and discussion of the results. Roberto Boada con- resulted in Se mainly found as selenoamino acids SeMet, SeCyst and a tributed to XAS data interpretation, manuscript writing and discussion small quantity of MeSeCys. The fact that the translocation of inorganic of the results. Tingting Xiao and Mercè Llugany contributed to hor- Se to the reproductive organs is minimal is advantageous to produce mone and mineral nutrient analysis. Manuel Valiente and Mercè Llu- fortified food products since inorganic Se is more toxic than its organic gany contributed to the original idea, research design and discussion counterparts (Rayman et al., 2008). These findings agree with previous of the results. All authors read and approved the manuscript. studies on wheat, even though our conditions led to lower amounts of inorganic Se and a higher percentage of SeCyst (Di et al., 2023; Wang ACKNOWLEDG MENTS et al., 2022; Wang, Ali, et al., 2020). The present study has been developed with the financial support of Span- Furthermore, the present study has shown that the enrichment ish Ministry MINECO grant CHEMNEXUS (CTM2015-65414-C2-1-R). conditions influence the selenomethionine/selenocystine proportion. Maria Angels Subirana acknowledges the support of MINECO The different mechanisms for selenite and selenate discussed above through BES-2013-066639 PhD grant. Roberto Boada acknowl- cause the diversity in the production of the specific selenoamino acids. edges funding support from the European Union's Horizon 2020 Selenite treatments resulted in the accumulation of Se in the roots in research and innovation program under the Marie Skłodowska-Curie high concentrations, causing a phytotoxicity response and the genera- grant agreement No. 665919. Tingting Xiao acknowledges the grant tion of ROS (Freeman et al., 2010). ROS species create an oxidizing from China Scholarship Council (201608330235). The authors environment in plant tissues (Mostofa et al., 2017) that enhances Se-Se acknowledge the ESRF facility and E. Salas-Colera for beamtime bounds formation from selenol groups (Reich & Hondal, 2016), resulting No. 25-01-967 at BM25A SpLine and M. Resina and V. Verdugo for in the oxidation of selenocysteine residues to selenocystine in the grain. their help during the synchrotron experiment. They also acknowl- In contrast, selenate stored in leaf vacuoles is stable and harmless to edge the Scientific and Technological Centers of the University of the plant (Hopper & Parker, 1999). Therefore, it does not promote an Barcelona (CCiTUB), and R. Padilla from the Plant Physiology Group oxidizing environment, which results in methylated selenoamino acids (BABVE, UAB) for the ICP-OES analysis. in the grain in the form of C-Se-C, such as SeMet and MeSeCys. Alter- natively, the characteristic selenoamino acid production can also be DATA AVAILABILITY STAT EMEN T explained as a tolerance strategy. SeCys and SeMet can be non- The data supporting the findings of this study are available from the specifically incorporated into proteins in place of cysteine and methio- corresponding author, Mercè Llugany, and the first author, Maria nine (Freeman et al., 2006). The larger size, polarizability, and reactivity Angels Subirana, upon reasonable request. of Se (Reich & Hondal, 2016) lead to protein misfolding, which alters protein function and cellular biochemical reactions (Kolbert et al., 2016). OR CID This nonspecific incorporation of selenoamino acids into proteins might Mercè Llugany https://orcid.org/0000-0002-9940-0383 be a major cause of the harmful effect of Se on sensitive plants. The enhanced formation of SeCyst and MeSeCys, which cannot be incorpo- RE FE RE NCE S rated into proteins, can be a defense mechanism of the plant to coun- Aborode, F.A., Raab, A., Foster, S., Lombi, E., Maher, W., Krupp, E.M. et al. teract the toxicity caused by selenite enrichment. (2015) Selenopeptides and elemental selenium in Thunbergia alata The difference in the selenoamino acid content depending on the after exposure to selenite: quantification method for elemental sele- enrichment conditions can be exploited to enhance the health bene- nium. Metallomics, 7, 1056–1066. fits of the use of Se-biofortified wheat grains as a functional food. Alfthan, G., Eurola, M., Ekholm, P., Venäläinen, E.-R., Root, T., Korkalainen, K. et al. (2015) Effects of nationwide addition of selenium SeCyst, MeSeCys, and SeMet are metabolized differently by humans to fertilizers on foods, and animal and human health in Finland: from and, accordingly, contribute to disease prevention (Weekley & deficiency to optimal selenium status of the population. Journal of Harris, 2013). In particular, the opposite outcomes of the NPC and Trace Elements in Medicine and Biology, 31, 142–147. SELECT cancer trials have demonstrated the critical difference in the Aureli, F., Ouerdane, L., Bierla, K., Szpunar, J., Prakash, N.T. & Cubadda, F. (2012) Identification of selenosugars and other low-molecular weight antineoplastic activity of the selenium compounds (Weekley & selenium metabolites in high-selenium cereal crops. Metallomics, 4, Harris, 2013). Both SeCyst and MeSeCys (Chen et al., 2019; 968–978. Weekley & Harris, 2013) are more active species than SeMet, Bañuelos, G.S., Freeman, J.L. & Arroyo, I.S. 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