92 93 D I A M O N D L I G H T S O U R C E A N N U A L R E V I E W 2 0 2 1 / 2 2 D I A M O N D L I G H T S O U R C E A N N U A L R E V I E W 2 0 2 1 / 2 2 The impact of applying a biostimulant on seleniumenrichedwheat Related publication: Xiao, T., Boada, R., Llugany, M., &Valiente, M. Co-application of Se and a biostimulant at different wheat growth stages: Influence on grain development. Plant Physiology and Biochemistry , 160 184–192 (2021). DOI: 10.1016/j.plaphy.2021.01.025 Publication keywords: Wheat biofortification; Se speciation; Functional food; Plant biostimulant S elenium is an essential nutrient for human health. The selenium-biofortification of crops is increasingly common in selenium-deficient regions. However, issues remain regarding the observed toxicity to the plant. Using a plant biostimulant can significantly reduce the stress experienced by wheat plants during selenium-biofortification processes. However, littlewas known regardingtheeffect of theplantbiostimulant over the seleniummetabolisationpathways, as theplant transforms inorganic selenium into more bioavailable organic forms (seleno-amino acids). To assess the success of combining the plant biostimulant treatmentwith the selenium-biofortificationprocess, researchers investigated the chemical species of seleniumpresent in the wheat grains (i.e., the ratio of the seleno-amino acids). The Microfocus Spectroscopy beamline (I18) offered the possibility of getting information, at themicroscopic level, about the distribution of the elements and the chemical species present on the wheat grain section. Their study proves that the biostimulant has a key role in enhancing both the number of grains produced per spike and their dry biomass without hindering the seleniumenrichment process, either by diminishing the seleniumconcentration ormassively disrupting the selenium species present in the wheat grain. Based on the successful results on wheat plants, similar enrichment studies are envisaged on other vegetal systems (e.g., alfalfa). The team aims to better understand the spatial distribution of selenium-bioavailable species and the toxic effects of selenium for the plant upon different biofortification conditions. This information will be useful to minimise both plant toxicity and economic cost and move towards more effective and plant-healthy selenium supplementation. Selenium (Se) is an essential trace mineral of fundamental importance to human health. In terms of the human metabolism, Se substitutes sulphur (S) in the amino acid groups forming antioxidant enzymes such as glutathione peroxidase (GPx), thioredoxin reductase (TrxR) and iodothyronine deiodinase (IDD) which are important, among other things, for protecting against oxidative stress and for regulating the thyroid hormone metabolism. Se, as many other nutrients, is introduced into the human body via food intake. For instance, food derived from plants is a natural source of Se since plants can transform inorganic Se species (e.g., selenium salts) present in the soil into organic ones (e.g., seleno-amino acids) which are the desired chemical form of Se for human diet since they are much more bioavailable than inorganic ones. Thus, Se level in the soil usually has a direct influence in the concentration of Se present in food and, subsequently, in the Se level found in the human body. Hence, regions with low Se level in soils would yield Se deficient diets. This is a major issue nowadays since up to 14% of the population worldwide suffers from having an inadequate dietary Se intake with the associated risk of developing several chronic degenerative diseases 1 . To achieve the appropriate Se level in the body, Se supplementation has been extensively used. For instance, soil fertilisation with Se has been applied in Finland since 1984 to increase Se concentration of food in regions with Se-deficient soils. However, the presence of high concentration of Se in soil induces stress to the plant and may hamper its normal development 2 . To overcome this issue, genetic engineering has been proposed as a strategy to enhance Se accumulation, volatilisation and/or tolerance in plants 3 . However, this approach has serious potential risks since it might promote the presence of new allergens in food, and the accumulation of other undesired heavy metals in the plant 4 . Alternatively, the application of a plant biostimulant as anti-stressor to alleviate the Se-induced toxicity in the plants emerges as a promising solution. In our work, we used wheat as a model crop since it is a widely used food for human food and livestock feed. The plants were grown on hydroponic cultivation to set the basis for future studies on soil cultures. We applied three different Se treatments (sodium selenite, Se(IV); sodium selenate, Se(VI); or a 1:1 mixture of both, Se(MIX)) together with the biostimulant (Fyto-fitness by BIO Fitos). The biostimulant was applied at two growing stages, tillering (when the plant has three or four leaves) or heading (when the spike begins to emerge), until harvesting the grains once matured. The total Se levels found ingrains for the different treatments indicated that Se-biofortification of grains was achieved with values within the range of 37– 100 μg.g -1 and of 75–138 μg.g -1 for heading and tillering stages, respectively. In addition, our results showed that the biostimulant have a key role increasing both the number of grains produced per spike and their biomass without diminishing the total amount of Se achieved by the biofortification processes. When the Se treatment is applied at the heading stage, the Se toxicity is less severe than when applied at the tillering stage. This can be attributed to the longer duration of the Se treatment in the case of the tillering stage. In that respect, the elemental mapping performed at beamline I18 5 via micro X-ray Fluorescence (µXRF) imaging allowed us to get detailed information about the Se distribution in the grain (Fig. 1). The maps showed that Se is unevenly distributed in the grain (warmer colours indicate higher Se concentration). The higher concentrations of Se are mostly found in the germ and outer layer regardless of the treatment applied. This is related to the fact that the outer layer, mostly the aleurone and the germ, are the main regions containing proteins and, therefore, the Se-proteins assembled from seleno- aminoacids are in those regions. Regarding the co-location with other micro and macronutrients, the analysis of the μXRF maps for the different elements indicated that aleurone and scutellum are major storage tissues for macro (e.g., P, K, Ca and Mg) as well as micro (e.g., Fe, Zn, Cu and Mn) nutrients (Fig. 2). This distribution does not get affected by either Se species supplied in the treatment or the application of plant biostimulant at either of the two different growth stages considered. However, it is not the amount of the Se accumulated but the chemical state of the Sewhich drives its bioavailability for humans. Therefore, determining the chemical state of Se is of crucial importance to assess the health benefits of the biofortification procedure. The chemical speciation analysis of Se performed by collecting micro X-ray Absorption Near-Edge Structure (μXANES) spectra at the regions of interest showed that the amount of organic Se species was always larger than 90%when the treatment was applied at the tillering stage, whereas for heading stage they were lower than 80% in most of the cases. The effect found in the grain is that the total Se content decreases together with the total organic Se, and that there is an increase of C–Se–C (e.g., selenomethionine) respect to the total organic Se found in the heading group. Although both C–Se–C and selenocysteine species are seleno-amino acids that can be incorporated into proteins in place of methionine and cysteine, leading to toxicity, C–Se–C species have less harmful effects. Indeed, elemental Se, Se(0), is only detected in the heading stage group of grains and it is negligible in the tillering ones. Se(0) is one of the products derived from SeCys via the action of a selenocysteine lyase (SL). Elemental Se is comparatively innocuous for the plant, therefore this could be a potential Se detoxification mechanism by the plant. In the heading group, the abiotic stress caused by Se when the grain spike is just appearing may stimulate the expression of SL to enhance Se tolerance and maintain the growth cycle. These results confirmed that the application of the plant biostimulant does not affect either the Se distribution or the Se speciation in the wheat grain and that the Se-biofortification from the heading stage is less toxic to the wheat plant than applications at earlier stages (e.g., tillering).] References: 1. Rayman, M. P. The importance of selenium to human health. The Lancet , 356 233–241(2000). DOI: 10.1016/S0140-6736(00)02490-9 2. Guerrero, B. et al . Dual effects of different selenium species on wheat. Plant Physiology and Biochemistry , 83 300–307 (2014). DOI: 10.1016/j. plaphy.2014.08.009 3. Progress in botany. Nature , 194 1023–1023 (1962). DOI: 10.1038/1941023a0 4. Buchanan, B. B. Genetic engineering and the allergy issue. Plant Physiology , 126 5–7 (2001). DOI: 10.1104/pp.126.1.5 5. Mosselmans, J. F. W. et al . I18 – the microfocus spectroscopy beamline at the Diamond Light Source. Journal of Synchrotron Radiation , 16 818–824 (2009). DOI: 10.1107/S0909049509032282 Funding acknowledgement: This research was supported by the Spanish CTM2015-65414-C2-1-R project from MINECO. We acknowledge Diamond Light Source beamtime no. SP18671-1 at I18 beamline. R. Boada acknowledges funding support from the European Union’s Horizon 2020 research and innovation program under the Marie Skłodowska-Curie grant agreement No. 665919. T. Xiao acknowledges the grant from China Scholarship Council (201608330235). Corresponding authors: Merc Llugany, Universitat Autònoma de Barcelona,
[email protected] Roberto Boada, Universitat Autònoma de Barcelona,
[email protected] Spectroscopy Group Beamline I18 Figure 1: X-ray fluorescence mapping of Se in wheat grains grown under different treatments applied at heading growth stage: No Biostimulant-NB and Foliar Application-FA, in combination with the Se treatments described in the text (sodium selenite, Se(IV); sodium selenate, Se(VI); or a 1:1 mixture of both, Se(MIX)). Figure 2: Optical image (a) and normalised μXRF elemental maps for Ca (b), Se (c) and Zn (d) of the wheat grain section for Se(VI) treatment applied at heading stage. Warmer colours indicate higher element concentration. Tricolour map image displaying the colocation of Ca, Se and Zn (e) and zoom on the bran region of the wheat grain (f ). Horizontal scale bar in panel (a) denotes 1mm. Figure 3: Results from the Se speciation analysis performed with the μXANES spectra collected at different parts of the wheat grain for biostimulant treatment applied at heading stage.