Effects of selenate and red Se-nanoparticles on the photosynthetic apparatus of Nicotiana tabacum.

Selenium (Se) is a natural trace element, which shifts its action in a relatively narrow concentration range from nutritional role to toxicity. Although it has been well established that in plants chloroplasts are among the primary targets, the mechanism of toxicity on photosynthesis is not well understood. Here, we compared selenate and red-allotrope elemental selenium nanoparticles (red nanoSe) in in vitro tobacco cultures to investigate their effects on the structure and functions of the photosynthetic machinery. Selenate at 10 mg/L concentration retarded plant growth; it also led to a decreased chlorophyll content, accompanied with an increase in the carotenoid-to-chlorophyll ratio. Structural examinations of the photosynthetic machinery, using electron microscopy, small-angle neutron scattering and circular dichroism spectroscopy, revealed significant perturbation in the macro-organization of the pigment-protein complexes and sizeable shrinkage in the repeat distance of granum thylakoid membranes. As shown by chlorophyll a fluorescence transient measurements, these changes in the ultrastructure were associated with a significantly diminished photosystem II activity and a reduced performance of the photosynthetic electron transport, and an enhanced capability of non-photochemical quenching. These changes in the structure and function of the photosynthetic apparatus explain, at least in part, the retarded growth of plantlets in the presence of 10 mg/L selenate. In contrast, red nanoSe, even at 100 mg/L and selenate at 1 mg/L, exerted no negative effect on the growth of plantlets and affected only marginally the thylakoid membrane ultrastructure and the photosynthetic functions.

Selenoamino Acid-Enriched Green Pea as a Value-Added Plant Protein Source for Humans and Livestock.

Selenium deficiency in various degrees affects around 15% of the world's population, contributing to a variety of health problems. In this study, we examined the accumulation and biotransformation of soil applied Se-supplementation (sodium selenite and sodium selenate forms) at different concentrations, along with growth and yield formation of green pea, in a greenhouse experiment. Biotransformation of inorganic Se was evaluated using HPLC-ICP-MS for Se-species separation in the above ground parts of green pea. Results showed 3 mg kg-1 SeIV increased green pea growth biomarkers and also caused an increase in protein content in leaves by 17%. Selenomethionine represented 65% of the total selenium content in shoots, but was lower in pods and seeds (54 and 38%, respectively). Selenomethionine was the major species in all plant parts and the only organic selenium form in the lower SeIV concentration range. Elevating the dose of SeIV (≥30 mg kg-1) triggered detrimental effects on growth and protein content and caused higher accumulation of inorganic Se in forms of SeVI and SeIV. Selenocysteine, another organic form of proteinogenic amino acid, was determined when SeIV (≥10 mg kg-1) was applied in higher concentrations. Thus, agronomic biofortification using the appropriate chemical form and concentration of Se will have positive effects on green pea growth and its enriched shoots and seeds provide a value-added protein source for livestock and humans with significant increased selenomethionine.

Biological changes of green pea (Pisum sativum L.) by selenium enrichment.

Supplement of common fertilizers with selenium (Se) for crop production will be an effective way to produce selenium-rich food and feed. The value of green pea seeds and forages as alternative protein source can be improved by using agronomic biofortification. Therefore, biological changes of green pea (Pisum sativum L.) and influences of inorganic forms of Se (sodium selenite and sodium selenate) at different concentrations on the accumulation of magnesium (Mg) and phosphorus (P) were investigated in greenhouse experiment. 3 mg kg-1 of selenite had positive effects to enhance photosynthetic attributes and decrease lipid peroxidation significantly. At the same time, Se accumulation increased in all parts of plant by increasing Se supply. Moreover, Mg and P accumulations were significantly increased at 3 mg kg-1 selenite and 1 mg kg-1 selenate treatments, respectively. By contrast higher selenite concentrations (≥30 mg kg-1) exerted toxic effects on plants. Relative chlorophyll content, actual photochemical efficiency of PSII (ФPSII) and Mg accumulation showed significant decrease while membrane lipid peroxidation increased. Thus, the present findings prove Se biofortification has positive effects on biological traits of green pea to provide it as a proper functional product.

Water-soluble organic selenometabolites of alfalfa (Medicago sativa L.) green biomass-derived fractions.

BACKGROUND: Tolerance of plants towards selenium, a non-essential microelement for higher plants, is a key issue when designing either the indirect (selenium-depletion from highly seleniferous soils) or directed (selenized feed production) enrichment of selenium in forages. Alfalfa (Medicago sativa L.), the well-known forage crop of the Fabaceae family, has been gaining considerable interest due to its application as a green manure, as a cover crop, or in soil remediation by nitrogen fixation. OBJECTIVE: The goal of our study was to assess into which selenocompounds alfalfa plants biotransform the excess selenium uptake from the soil. Selenocompounds (other than selenomethionine and inorganic forms) accumulated in the fiber and the so-called brown juice by-product fractions of processed alfalfa biomass were targeted. METHODOLOGY: Inductively coupled plasma - mass spectrometry assisted multidimensional (strong anion exchange, strong cation exchange, reversed phase) orthogonal chromatographic purification was applied to supply Se-containing fractions in adequately high purity for electrospray high-resolution mass spectrometry (used for the first time for this matrix) analyses. RESULTS: As a total, 30 selenocompounds (with isomers) were described, showing the abundance of the derivatives of selenohexose, selenohomolanthionine, and 2,3-dihydroxypropionic acid. Out of the 30 selenocompounds, 15 could be assigned the elemental composition, and the tentative structure of five compounds including among others deamino-2-oxo-selenohomolanthionine, deamino-hydroxy-selenohomolanthionine, and the dimer of 2,3-dihydroxypropionyl-selenohomocysteine could be presented. CONCLUSIONS: The studied fractions arising from the standard alfalfa processing technology contained a wide variety of selenocompounds whose origin can be either the plant's inherent Se metabolism or the processing technology itself. The importance of negative mode data acquisition has been highlighted, as out of the 30 compounds, 16 could be detected exclusively in this electrospray ionization mode.

Living mulch enhances soil enzyme activities, nitrogen pools and water retention in giant reed (Arundo donax L.) plantations.

Giant reed (Arundo donax L.) is one of the most well-studied perennial biomass crops because of its high productivity and potential to store carbon. Yet, little information on controlling weeds in giant reed plantations and their influences on the soil ecosystem is available. In the present study, three different weed control methods, i.e., intercropping (living mulch) with sweet clover (Melilotus officinalis L.), herbicide (glyphosate), and hoeing, were investigated in a 2-year giant reed farm. The intercropping presented significantly higher values (on average) of all the tested soil properties than herbicide and hoeing, except for the catalase activity and pH. The dehydrogenase, phosphatase, and urease activities in the soil under intercropping were higher than the herbicide by 75%, 65%, and 80% (on average), respectively. Also, the soil under intercropping had higher soil organic matter (SOM) and soil respiration than the herbicide by 20% and 25%, respectively. Intercropping also increased the content of N pools, i.e., NO3--N, NH4+-N, Org-N, and Total-N by 517%, 356%, 38%, and 137%, respectively, compared to herbicide. These findings illustrated that controlling weeds in biomass plantations through legume intercropping brings benefits not only to soil properties but also to biomass productivity.

Green Biomass-Based Protein for Sustainable Feed and Food Supply: An Overview of Current and Future Prospective.

It is necessary to develop and deploy novel protein production to allow the establishment of a sustainable supply for both humans and animals, given the ongoing expansion of protein demand to meet the future needs of the increased world population and high living standards. In addition to plant seeds, green biomass from dedicated crops or green agricultural waste is also available as an alternative source to fulfill the protein and nutrient needs of humans and animals. The development of extraction and precipitation methods (such as microwave coagulation) for chloroplast and cytoplasmic proteins, which constitute the bulk of leaf protein, will allow the production of leaf protein concentrates (LPC) and protein isolates (LPI). Obtained LPC serves as a sustainable alternative source of animal-based protein besides being an important source of many vital phytochemicals, including vitamins and substances with nutritional and pharmacological effects. Along with it, the production of LPC, directly or indirectly, supports sustainability and circular economy concepts. However, the quantity and quality of LPC largely depend on several factors, including plant species, extraction and precipitation techniques, harvest time, and growing season. This paper provides an overview of the history of green biomass-derived protein from the early green fodder mill concept by Károly Ereky to the state-of-art of green-based protein utilization. It highlights potential approaches for enhancing LPC production, including dedicated plant species, associated extraction methods, selection of optimal technologies, and best combination approaches for improving leaf protein isolation.

Comparison of Wet Fractionation Methods for Processing Broccoli Agricultural Wastes and Evaluation of the Nutri-Chemical Values of Obtained Products.

The main objective of this study was to increase the economic value of broccoli green agro-waste using three wet fractionation methods in the shadow of green biorefinery and the circular economy. Product candidates were obtained directly by using a mechanical press, and indirectly by using microwave coagulation or via lactic acid fermentation of green juice. The leaf protein concentrates (LPC) fractions displayed significantly higher dry matter content and crude protein content (34-39 m/m% on average) than the green juice fraction (27.4 m/m% on average), without considerable changes in the amino acids composition ratio. UHPLC-ESI-ORBITRAP-MS/MS analysis showed that kaemferol and quercetin are the most abundant flavonols, forming complexes with glycosides and hydroxycinnamic acids in green juice. Lacto-ermentation induced a considerable increase in the quantity of quercetin (48.75 µg·g-1 dry weight) and kaempferol aglycons (895.26 µg·g-1 dry weight) of LPC. In contrast, chlorogenic acid isomers and sulforaphane disappeared from LPC after lactic acid fermentation, while microwave treatment did not cause significant differences. These results confirm that both microwave treatment and lacto-fermentation coagulate and concentrate most of the soluble proteins. Also, these two processes affect the amount of valuable phytochemicals differently, so it should be considered when setting the goals.

Bioactive metabolite profile and antioxidant properties of brown juice, a processed alfalfa (Medicago sativa) by-product.

Recently, leaf protein concentrate (LPC) has gained increased attention in response to the constantly growing protein demand. Green biorefineries can become more economical by valorizing their by-products and reducing environmental risks. The current study describes the variations in the antioxidant capacity and phytochemical composition of a liquid by-product (referred to as brown juice (BJ)) obtained during the extraction of leaf protein concentrate (LPC) from the fresh biomass of alfalfa (Medicago sativa L.). Four varieties of alfalfa were investigated during three harvest times, i.e., August 2017 (first harvest), September 2017 (second harvest), and June 2018 (third harvest). Also, the fresh BJ was lacto-fermented to extend its preservation period but also modifying its composition. The results of different general phytochemical composition analyses and antioxidant assays revealed similar tendencies across different alfalfa varieties and harvest times. Most of the phytochemicals in the BJ identified by HPLC-MS/MS can be classified as flavonoids/flavonoid derivatives, e.g., apigenin, naringenin, luteolin, formononetin. Substantially, the lacto-fermentation process induced a switch into aglycones, e.g., apigenin content increased by an order of magnitude, while apigenin-7-O-glucuronide content was halved after lacto-fermentation. Additionally, several B vitamins were detected, including B2, B3, and B7. These results could provide a basis for various ways of industrial valorization but need to be strengthened by data generated from large-scale production.

Uptake Dynamics of Ionic and Elemental Selenium Forms and Their Metabolism in Multiple-Harvested Alfalfa (Medicago sativa L.).

A pot experiment, under greenhouse conditions, was carried out aiming at investigating the agronomic biofortification of alfalfa (Medicago sativa L.) with Se and monitoring the Se uptake and accumulation dynamics within four consecutive harvests within the same growing season. Two ionic Se forms, i.e., sodium selenate (Se (VI)) and sodium selenite (Se (IV)), were applied once at a rate of 1, 10, and 50 mg kg-1 (added on Se basis), while 10 and 50 mg L-1 of a red elemental Se (red Se0) were used; all Se treatments were added as soil application. Application of Se (VI) at the rate of 50 mg kg-1 was toxic to alfalfa plants. The effect of Se forms on Se accumulation in alfalfa tissues, regardless of the applied Se concentration, follows: Se (VI) > Se (IV) > red Se0. The leaf, in general, possessed higher total Se content than the stem in all the treatments. The accumulation of Se in stem and leaf tissues showed a gradual decline between the harvests, especially for plants treated with either Se (VI) or Se (IV); however, the chemically synthesized red Se0 showed different results. The treatment of 10 mg kg-1 Se (VI) resulted in the highest total Se content in stem (202.5 and 98.0 µg g-1) and leaf (643.4 and 284.5 µg g-1) in the 1st and 2nd harvests, respectively. Similar tendency is reported for the Se (IV)-treated plants. Otherwise, the application of red Se0 resulted in a lower Se uptake; however, less fluctuation in total Se content between the four harvests was noticed compared to the ionic Se forms. The Se forms in stem and leaf of alfalfa extracted by water and subsequently by protease XIV enzyme were measured by strong anion exchange (SAX) HPLC-ICP-MS. The major Se forms in our samples were selenomethionine (SeMet) and Se (VI), while neither selenocysteine (SeCys) nor Se (IV) was detected. In water extract, however, Se (VI) was the major Se form, while SeMet was the predominant form in the enzyme extract. Yet, Se (VI) and SeMet contents declined within the harvests, except in stem of plants treated with 50 mg L-1 red Se0. The highest stem or leaf SeMet yield %, in all harvests, corresponded to the treatment of 50 mg L-1 red Se0. For instance, 63.6% (in stem) and 38.0% (in leaf) were calculated for SeMet yield % in the 4th harvest of plants treated with 50 mg L-1 red Se0. Our results provide information about uptake and accumulation dynamics of different ionic Se forms in case of multiple-harvested alfalfa, which, besides being a good model plant, is an important target plant species in green biorefining.

Identification of Bioactive Phytochemicals in Leaf Protein Concentrate of Jerusalem Artichoke (Helianthus tuberosus L.).

Jerusalem artichoke (JA) is widely known to have inulin-rich tubers. However, its fresh aerial biomass produces significant levels of leaf protein and economic bioactive phytochemicals. We have characterized leaf protein concentrate (JAPC) isolated from green biomass of three Jerusalem artichoke clones, Alba, Fuseau, and Kalevala, and its nutritional value for the human diet or animal feeding. The JAPC yield varied from 28.6 to 31.2 g DM kg-1 green biomass with an average total protein content of 33.3% on a dry mass basis. The qualitative analysis of the phytochemical composition of JAPC was performed by ultra-high performance liquid chromatography-electrospray ionization-Orbitrap/mass spectrometry analysis (UHPLC-ESI-ORBITRAP-MS/MS). Fifty-three phytochemicals were successfully identified in JAPC. In addition to the phenolic acids (especially mono- and di-hydroxycinnamic acid esters of quinic acids) several medically important hydroxylated methoxyflavones, i.e., dimethoxy-tetrahydroxyflavone, dihydroxy-methoxyflavone, hymenoxin, and nevadensin, were detected in the JAPC for the first time. Liquiritigenin, an estrogenic-like flavanone, was measured in the JAPC as well as butein and kukulkanin B, as chalcones. The results also showed high contents of the essential amino acids and polyunsaturated fatty acids (PUFAs; 66-68%) in JAPC. Linolenic acid represented 39-43% of the total lipid content; moreover, the ratio between ω-6 and ω-3 fatty acids in the JAPC was ~0.6:1. Comparing the JA clones, no major differences in phytochemicals, fatty acid, or amino acid compositions were observed. This paper confirms the economic and nutritional value of JAPC as it is not only an alternative plant protein source but also as a good source of biological valuable phytochemicals.

Selenate tolerance and selenium hyperaccumulation in the monocot giant reed (Arundo donax), a biomass crop plant with phytoremediation potential.

The response of giant reed (Arundo donax L.) to selenium (Se), added as selenate, was studied. The development, stress response, uptake, translocation, and accumulation of Se were documented in three giant reed ecotypes STM (Hungary), BL (USA), and ESP (Spain), representing different climatic zones. Plantlets regenerated from sterile tissue cultures were grown under greenhouse conditions in sand supplemented with 0, 2.5, 5, and 10 mg Se kg-1 added as sodium selenate. Total Se content was measured in different plant parts using hydride generation atomic fluorescence spectroscopy. All plants developed normally in the 0-5.0 mg Se kg-1 concentration range regardless of ecotype, but no growth occurred at 10.0 mg Se kg-1. There were no signs of chlorosis or necrosis, and the photosynthetic machinery was not affected as evidenced by no marked differences in the structure of thylakoid membranes. There was no change in the maximum quantum yield of photosystem II (Fv/Fm ratio) in the three ecotypes under Se stress, except for a significant negative effect in the ESP ecotype in the 5.0 mg Se kg-1 treatment. Glutathione peroxidase (GPx) activity increased as the Se concentration increased in the growth medium. GPx activity was higher in the shoot system than the root system in all Se treatments. All ecotypes showed great capacity of take up, translocate and accumulate selenium in their stem and leaf. Relative Se accumulation is best described as leaf ˃˃ stem ˃ root. The ESP ecotype accumulated 1783 µg g-1 in leaf, followed by BL with 1769 µg g-1, and STM with 1606 µg g-1 in the 5.0 mg Se kg-1 treatment. All ecotypes showed high values of translocation and bioaccumulation factors, particularly the ESP ecotype (10.1 and 689, respectively, at the highest tolerated Se supplementation level). Based on these findings, Arundo donax has been identified as the first monocot hyperaccumulator of selenium, because Se concentration in the leaves of all three ecotypes, and also in the stem of the ESP ecotype, is higher than 0.1% (dry weight basis) under the conditions tested. Tolerance up to 5.0 mg Se kg-1 and the Se hyperaccumulation capacity make giant reed a promising tool for Se phytoremediation.