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The influence of exogenous methyl jasmonate on the germination and, content and composition of flavonoids in extracts from seedlings of yellow and narrow-leafed lupine
Journal of Food Composition and Analysis 87 (2020) 103398
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Original Research Article
The influence of exogenous methyl jasmonate on the germination and, content and composition of flavonoids in extracts from seedlings of yellow and narrow-leafed lupine
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Kazimierz Zalewskia, Bartosz Nitkiewicza,*, Mariusz Stolarskib, Ryszard Amarowiczc, Adam Okorskid a
Department of Biochemistry, Faculty of Biology and Biotechnology, University of Warmia and Mazury, Olsztyn, Poland Department of Plant Breeding and Seed Production, University of Warmia and Mazury, Olsztyn, Poland Division of Food Science, Institute of Animal Reproduction and Food Research of the Polish Academy of Sciences, Olsztyn, Poland d Department of Entomology, Phytopathology and Molecular Diagnostics, University of Warmia and Mazury, Olsztyn, Poland b c
A R T I C LE I N FO
A B S T R A C T
Keywords: Jasmonates JA-Me Flavonoids Lupine seedlings
The aim of this study was to determine the influence of exogenous methyl jasmonate (MJ) on the content and composition of flavonoids (isoflavones) in extracts from hypocotyls (with cotyledons) and radicles of yellow lupine (Lupinus luteus L.) var. Polo and in extracts from radicles of narrow-leafed lupine (Lupinus angustifolius) var. Graf. Lupine seeds were harvested when fully ripe. Two months after harvest, the effect of various MJ concentrations (10−6 M to 10-3M) on seed viability, seed vigor and the content and composition of flavonoids in extracts from seedlings that emerged from germinated lupine seeds (72 h, 20 °C) was determined. At high concentrations (10-4 M to 10-3 M), MJ suppressed the germination rate and germination capacity of seeds and decreased the growth rate of seedlings of the analyzed varieties of yellow and narrow-leafed lupines in the first 5 days of growth. In seedlings, MJ significantly increased the content of isoflavones (including daidzin, genistin, daidzein, and genistein) in 3-day-old hypocotyls (with cotyledons) and radicles of yellow lupine. This correlation was also observed in the hypocotyls (with cotyledons) and radicles of 3-day-old narrow-leafed lupine seedlings treated with MJ. Narrow-leafed lupine seeds were more sensitive to exogenous MJ then yellow lupine seeds during germination.
1. Introduction The structures and biosynthetic pathways of jasmonates, mainly jasmonic acid (JA) and methyl jasmonate (MJ), are similar to those of prostaglandins found in animals. Jasmonates have various effects and are capable of inducing (microtubule decomposition, phytoalexin and alkaloid synthesis, tuber formation, shoot shortening), stimulating (aging, chlorophyll degradation, respiration, and leaf shedding) and inhibiting (Rubisco synthesis, photosynthesis, cell division, embryogenesis, flowering, and seed germination) various biological processes (Sembdner and Parthier, 1993). Exogenous MJ modifies the activity of α-D-galactosidase and the composition of carbohydrates in yellow lupine seedlings (Zalewski et al., 2010). Foliar-applied MJ changes the expression of genes encoding fruit ripening, pollen production, shoot
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growth, root hair growth and resistance to pathogens (Creelman and Mullet, 1997). Jasmonates play an important role in the production of secondary metabolites, including flavonoids. Mechanical injury, pest damage and fungal infections lead to the release of linolenic acid from membrane lipids. Linolenic acid is converted to JA in the octadecanoid pathway. In cells, higher concentrations of JA influence the expression of various genes involved in defense. Jasmonic acid also stimulates anthocyanin biosynthesis (Grzesiuk et al., 2008). Jasmonates resemble abscisic acid in many respects, in particular with regard to plant responses to stress (Parthier, 1991). Jasmonic acid and MJ are the key signaling compounds that induce the expression of genes encoding inhibitors of proteinases (proteins that protect plants against insects) in response to plant damage (Farmer and Ryan, 1990). Methyl jasmonate increases the transcription of genes encoding defensins (PDE 1.2) and
Corresponding author at: Department of Biochemistry, Faculty of Biology and Biotechnology, University of Warmia and Mazury, Oczapowskiego 1A, 10-719, Olsztyn, Poland. E-mail addresses: [email protected] (K. Zalewski), [email protected] (B. Nitkiewicz), [email protected] (M. Stolarski), [email protected] (R. Amarowicz), [email protected] (A. Okorski). https://doi.org/10.1016/j.jfca.2019.103398 Received 16 July 2019; Accepted 27 December 2019 Available online 28 December 2019 0889-1575/ © 2019 Elsevier Inc. All rights reserved.
Journal of Food Composition and Analysis 87 (2020) 103398
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Table 1 Viability and vigor of yellow lupine (var. Polo) and narrow-leafed lupine (var. Graf) seeds. Standard deviation is expressed as the number of significant digits. Combination
Germination rate (10 days for yellow lupine and 5 days for narrow-leafed lupine, 20 °C) (%)
Yellow lupine (Lupinus luteus) Control 81.50 ± 1.2 MJ x 10 −6M 81.00 ± 3.1 −5 MJ x 10 M 79.00 ± 3.3 MJ x 10 −4M 64.50 ± 4.0 −3 M 42.25 ± 2.1 MJ x 10 Narrow-leafed lupine (Lupinus angustifolius) Control 75.50 ± 2.4 MJ x 10 −6M 76.00 ± 3.6 MJ x 10 −5M 63.50 ± 3.0 MJ x 10 −4M 52.25 ± 2.1 MJ x 10 −3M 34.50 ± 1.9
Germination capacity (21 days for yellow lupine and 10 days for narrowleafed lupine, 20 °C) (%)
Seedling growth analysis (germination 5 days, 20 °C) Primary root lenght (mm)
Hypocotyl lenght (mm)
Fresh weight of root (g)
Fresh weight of hypocotyl with cotyledons (g)
94.50 ± 2.0 95.00 ± 2.5 91.50 ± 2.5 82.50 ± 2.0 57.00 ± 0.7
42.23 ± 2.5 38.62 ± 0.5 22.70 ± 1.8 15.61 ± 2.1 12.35 ± 0.6
35.2 ± 2.8 34.9 ± 2.9 26.3 ± 1.9 23.5 ± 2.2 12.7 ± 2.3
1.73 ± 0.22 1.67 ± 0.34 1.45 ± 0.20 1.32 ± 0.13 1.13 ± 0.08
5.71 ± 0.26 5.50 ± 0.33 4.36 ± 0.30 3.98 ± 0.24 3.34 ± 0.31
92.25 ± 4.2 93.00 ± 4.3 81.25 ± 4.5 73.50 ± 3.5 42.50 ± 2.9
29.82 ± 2.1 28.33 ± 2.3 22.39 ± 2.0 16.51 ± 1.8 5.10 ± 1.6
34.03 ± 2.8 34.71 ± 2.9 27.43 ± 3.1 15.90 ± 2.5 4.41 ± 1.0
1.07 ± 0.11 1.08 ± 0.10 0.86 ± 0.09 0.56 ± 0.05 0.11 ± 0.03
5.25 ± 0.23 5.17 ± 0.34 4.21 ± 0.41 3.38 ± 0.27 3.13 ± 0.11
Company, St. Paul, USA). Germination rates of yellow and narrowleafed lupine seeds were determined after 10 and 5 days of incubation, respectively (International Seed Testing Associacion (ISTA), 1996). Organic solvents were purchased from Merck (Warsaw, Poland). Trifluoroacetic acid was obtained from Sigma-Aldrich (Poznań, Poland). Standards of daidzin, daidzein, genistin, genistein, and apigenin were purchased from Extrasynthese (Genay Cedex, France).
thionins (THI 2.1), proteins with antibacterial properties (Farmer and Ryan, 1990; Szczegielniak, 2006). Jasmonic acid-dependent signaling pathways activate systemic responses, whereas JA-independent signaling pathways are involved in tissue repair and pathogen defense (Szczegielniak, 2006). According to (Fonseca et al., 2009), jasmonates regulate plant development and adaptation to adverse environmental conditions by deactivating selected natural plant hormones. When applied directly to leaves, JA increases the total phenolic content of basil plants. As an abiotic elicitor, JA could be used to enhance the biological activity and health benefits of sweet basil landraces (Malekpoor et al., 2016). The resistance of tomato seedlings to Fusarium oxysporum f. sp. lycopersici increased 4 weeks after inoculation when tomato seeds had been immersed in a solution of 0.1 mM methyl jasmonate for 1 h. At concentrations of 0.01, 0.1 and 1 mM, MJ inhibited spore germination and the growth of the fungal strain in vitro (Król et al., 2015). The MJ-induced increase in the content of phenolic compounds in plants delivers health benefits to consumers. The positive influence of jasmonates on reducing the development of some human cancer diseases has already been proven (Majewska-Wierzbicka et al., 2012; Yach et al., 2004). Research indicates that jasmonates reduce the risk of certain cancers in humans (Majewska-Wierzbicka et al., 2012). Methyl jasmonate inhibits the proliferation of neuroma and prostate cancer cells, and it significantly decreases the risk of prostate cancer (Tong et al., 2008). Spraying friut with MJ before harvest has positive health implications for consumers (Reyes-Díaz et al., 2016). There is growing evidence to indicate that this plant hormone has health benefits for humans and animals, therefore; it will continue to be a subject of debate in the future (Cohen and Flescher, 2009; Umukoro et al., 2011). Seeds with proteins as their basic material react more strongly than oilseeds eg. sunflower seeds (Białecka and Kępczyński, 2003). This was one of the reasons for using lupine seeds in experiments. In addition, several species of lupins are cultivated in Poland, but only eleven species are grown on the production fields. Three of them are grown for fodder purposes: yellow, narrow-leaved and white lupins. The growing area is about 40,000 ha in 2017 and around 60,000 ha in 2018.
2.1. Germination Lupine seeds were incubated in solutions of 10−3 M, 10-4 M, 10-5 M or 10-6 M MJ (20 °C). Seeds germinated in distilled water were served as the control. After 3 days of germination, hypocotyls with cotyledons and radicles were isolated from yellow lupine seedlings. For comparative purposes, only radicles were isolated from narrow-leafed lupine seedlings, and they were used in flavone and isoflavone analyses. 2.2. Seedling growth analysis Seedling growth was analyzed after 5 days of seed incubation (100 seeds in four replications) in distilled water in the dark at a temperature of 20 °C, different concentrations of the applied growth regulator. The following seedling parameters were analyzed: radicle length, hypocotyl length, fresh weight of hypocotyls with cotyledons, and fresh radicle weight. 2.3. Extraction of phenolic compounds Isolated hypocotyls with cotyledons and isolated radicles were frozen in liquid nitrogen and ground in an impact mill. Phenolic compounds were extracted in three replications of 15 min each, with 70% methanol, at 40 °C, in a Julabo SW-22 shaking water bath (WITKO, Warsaw, Poland). Methanol extracts were combined, and organic solvent was evaporated in a Büchi R-200 vacuum evaporator (Büchi, Warsaw, Poland) at 40 °C. The remaining water was removed by freeze drying (Labconco, Kansas City, MO, USA).
2. Materials and methods 2.4. HPLC analysis The study was carried out on yellow and narrow-leafed lupine seedlings. Seeds were harvested when fully ripe; they were then dried and stored in linen bags at 16−18 °C and a mean humidity of 60–65%. The average thousand seed weight (at 11% moisture content) was determined to be 105.2 g for yellow lupine and 143.4 g for narrow-leafed lupine. Germination capacity was determined as the percentage of yellow lupine seeds that germinated after 21 days of imbibition and the percentage of narrow-leafed lupine seeds that germinated after 10 days of imbibition on moistened germination paper (Anchor Paper
The obtained extract was dissolved in 70% methanol and passed through a 0.45 μm filter. The HPLC analysis was carried out in a Shimadzu HPLC system (Shimadzu, Kyoto, Japan) composed of two LC10AD pumps, an SPD-M 10A photodiode detector and an SCTL-10A controller. Phenolic compounds were separated on a Luna C18 column (4 x 250 mm, 5 μm; Phenomenex, Torrance, CA, USA) in a 5–40% aqueous solution of acetonitrile with the addition of 0.1% trifluoroacetic acid (TFA) for 50 min. The injection volume was 20 μl, the 2
Journal of Food Composition and Analysis 87 (2020) 103398
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Fig. 1. Chromatographic separation (HPLC) of isoflavones and flavones isolated from the radicles of narrow-leafed lupine seedlings. A – control; B –10−4M MJ; C-10M MJ.
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flow rate was 1 ml/min, and detection wavelength was 260 nm and 320 nm. For quantitative analysis four standards of isoflavones (daidzein, daidzin, genistein, genistin) and one standard of flavone (apigenin) were used. The external standard method for quantitative analysis. The linearity of calibration curves (standard peak area versus standard concentration) was characterized by correlation coefficients r > 0.995. Isoflavones were determined at 260 nm and flavones at 320 nm. The content of flavones was expressed as apigenin equivalents. All the chemical determinations were performed in triplicate. The results are reported as means ± SD (Table 1). Differences in the content of each isoflavones were statistically evaluated by ANOVA and Tukey’s posttest comparisons. Statistical calculations were performed using:
STATISTICA software ver. 10 (StatSoft, Inc.). 3. Results An analysis of lupine seed viability demonstrated that germination capacity exceeded 90% in both lupine species (control – germination in H2O). High concentrations of MJ (10−3 M, 10-4 M and partially 10-5 M) decreased germination rate and germination capacity. Narrow-leafed lupine seeds were more sensitive to exogenous MJ then yellow lupine seeds. An analysis of 5-day-old seedlings revealed similar results (Table 1). Methyl jasmonate exerted a more pronounced effect on yellow lupine radicles (smaller increase in length and lower fresh weight of radicles of hypocotyls). 3
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Fig. 2. UV–vis absorption spectra of isoflavones from lupine seedling extracts and the genistein standard. Numbers 1–4 correspond to the peaks in Fig. 1.
concentrations of 10−6 M to 10-5 M, and c) the highest concentrations of MJ applied during seed germination (10-4 M and, 10-3 M). All differences between the above groups were statistically significant. The content of compounds 1–4 was expressed in terms of the apigenin equivalent, and the content of compounds 7 and 8, – in terms of the genistein equivalent. For the control sample (germination in distilled water) the content of compounds 1–4 was expressed in terms of the genistin equivalent, and the content of compounds 5–8, – in terms of the apigenin equivalent. The extracts from the radicles of germinating narrow-leafed lupine seeds (control sample) contained 7 flavonoids, including 17.94 mg of isoflavones/g of the extract and 5.31 mg of flavones/g of the extract (Fig. 5). The presence of jasmonate in incubation mixtures induced changes in the flavonoid content of roots. In samples exposed to MJ at a concentration of 10−4 M, the flavonoid content of roots increased (genistin and compound no. 4) or decreased (compound no. 1, daidzin, daidzein and compound no. 8). Exposure to the highest jasmonate concentration significantly increased the content of the analyzed compounds relative to the control sample (Fig. 5), in particular compound 1 of the genistin family the content of which increased more than 5-fold. Daidzin and compound 4 were the predominant components in the group of 8 flavonoids identified in hypocotyls with cotyledons isolated from the radicles of yellow lupine seedlings (Table 2). In the control sample compound 7 (Table 2) predominated in the group of flavones. Only the content of genistin derivative (compound no. 1 in Table 2) did
The HPLC chromatograms of the analyzed extracts of phenolic compounds isolated from the radicles of narrow-leafed lupine seedlings (Fig. 1) revealed four main isoflavone peaks (1–4) registered at 260 nm (retention times: 23.8, 28.4, 29.5, and 33.9 min) and four flavone peaks (5–8) registered a 320 nm (retention times: 30.8, 31.5, 35.3, and 44.7 min) (Fig. 1). It should be noted that peak 6 was visible only in the chromatogram of the sample incubated with the highest concentration of MJ. The spectra of compounds corresponding to peaks 1–4 were characterized by the maximum absorption at 259–260 nm. The spectra of those compounds were identical to the genistein spectrum (Fig. 2). Two absorption bands were observed in the spectra of compounds corresponding to peaks 5–8. The maximum absorption was noted at 267 nm for the first band and at 324−336 nm for the second band. The spectra of those compounds were highly similar to the apigenin spectrum (Fig. 3). Seven flavonoids were identified in the radicles of germinating yellow lupine seeds, 4 of which were detected based on the simultaneous separation of the reference standards. The data presented in Fig. 4 indicate that the content of the analyzed compounds varied from 0.71 μg to more than 8 μg per mg under control conditions. All concentrations of exogenous MJ enhanced the biosynthesis of all flavonoids, and the synthesis of an apigenin group compound (no. 7 in Fig. 4) increased 14-fold. The results presented in Fig. 4 can be divided into three significantly different groups: a) control, b) MJ 4
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Fig. 3. UV–vis absorption spectra of flavones from lupine seedling extracts and the apigenin standard. Numbers 5–8 correspond to the peaks in Fig. 1. Fig. 4. Flavonoid content of methanol extracts from the radicles of germinating yellow lupine seeds (μg/mg of extract). Compounds: 1,4,7 expressed as apigenin equivalents; 2,5,6 expressed as genistin equivalents (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).
4. Discussion
not differ significantly between the experimental treatments. The content of the remaining flavonoids differed significantly at all concentrations of MJ relative to the control sample. The greatest increase was observed in daidzein content (more than 12-fold) in response to the highest concentration of MJ during seed incubation.
MJ causes less water accumulation in germinated seeds (cell wall lignification), limiting the growth of seedlings. Therefore, the content of flavonoids is presented as μg per mg of methanol extract obtained, which is common in many publications. The observed increase in the flavonoid content of lupine seedlings
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Fig. 5. The content of individual phenolic compounds in extracts from narrow-leafed lupine radicles (μg/mg of extract; the quantity of extracts was identical in all samples). Compounds: 1,4,7,8 expressed as apigenin equivalents; 2,5,6 expressed as genistin equivalents. Table 2 The flavonoid content of methanol extracts from the hypocotyls (with cotyledons) of germinating yellow lupine seeds (μg/mg of extract). Compounds 1,4,7,8 expressed as apigenin equivalents. Flavonoid derivative
Control
MJ 10−6 M
MJ 10−5 M
MJ 10−4 M
MJ 10−3 M
1 Daidzin Genistin 4 Daidzein Genistein 7 8 Together
1.75 ± 0.09 3.65 ± 0.02 2.41 ± 0.11 3.65 ± 0.13 0.26 ± 0.01 1.23 ± 0.07 4.36 ± 0.24 0.55 ± 0.03 17.98 ± 0.63
2.12 ± 0.11 6.16 ± 0.34 3.92 ± 0.18 6.85 ± 0.24 0.21 ± 0.01 2.25 ± 0.14 6.35 ± 0.35 0.72 ± 0.04 28.58 ± 1.26
1.97 ± 0.10 4.12 ± 0.23 4.06 ± 0.18 4.01 ± 0.14 0.89 ± 0.05 1.98 ± 0.12 5.10 ± 0.28 1.10 ± 0.06 23.23 ± 1.19
2.26 ± 0.11 9.08 ± 0.50 3.85 ± 0.17 10.35 ± 0.36 2.89 ± 0.15 3.14)5 ± 0.19 8.02 ± 0.45 0.83 ± 0.05 40.43 ± 2.23
1.85 ± 0.09 7.03 ± 0.39 3.75 ± 0.17 8.69 ± 0.60 3.20 ± 0.17 3.11 ± 0.19 8.36 ± 0.46 2.36 ± 0.13 38.35 ± 2.28
The mycotoxin content of seeds of fungi infested plants is another important consideration. Lupine seeds are processed into lupine flour, meat substitutes, plant milk, yogurt and cheese (Feldheim, 2000; Hill, 1986). Legume seeds are also widely used in the production of animal feeds. Members of the genus Fusarium are among the most pathogenic toxin-producing fungi that colonize lupine plants (Selwet, 2010). Not all seeds of fungi-infected plants contain mycotoxins. However, the use of contaminated seeds in food and feed production poses significant health risks (Zain, 2011). Mycotoxins are not degraded at temperatures below 250 °C and are not eliminated during cooking. Therefore, further research into effective methods of eliminating mycotoxins from foodstuffs and feedstuffs, including jasmonate treatments of seeds (pre-imbibition) and seedlings, is required, and its results should have direct practical applications. Flavonoids are a diverse group of phenolic compounds that occur naturally in plants. These secondary metabolites deliver health benefits (Cohen and Flescher, 2009; Ranilla et al., 2009; Reyes-Díaz et al., 2016). Apigenin is a naturally occurring flavonoid in parsley, thyme, chamomile and red pepper, and the biosynthesis of its derivatives in lupine seedlings is stimulated by exogenous MJ. Apigenin speeds up the formation of nerve cells from stem cells, and strengthens connections between brain cells, which can improve memory and learning (Paulsen et al., 2011). For these reasons, apigenin may have the potential to treat Parkinson’s disease and Alzheimer’s disease (Miller et al., 2008). Jasmonates have also been found to suppress cell proliferation and induce apoptosis in human and mouse breast cancer, prostate cancer, melanoma, lymphoblastic leukemia (Fingrut and Flescher, 2002) and lymphoma cell lines (Reischer et al., 2007). The anticarcinogenic effects of jasmonates have been described in a review article (Flescher, 2007). Further research is required to address two key issues: a) whether
treated with exogenous MJ should be analyzed from several points of view because research into flavonoids pursues several objectives. One of them is the influence of flavonoids on plant development and resistance to pathogens. Other lines of research investigate the health benefits of flavonoids and other phenols in plant-based foods (Ranilla et al., 2009). Attempts have also been made, in particular in Australia and New Zealand, to determine mycotoxin levels in lupine seeds which are used in the production of animal feeds and can pose a significant health threat, especially to sheep and pigs (Battilani et al., 2011). The current study, which investigated the influence of exogenous MJ on germinating lupine seeds, falls within the above mentioned research scope. Lupines are particularly susceptible to fungal infections which cause anthracnose and Fusarium diseases, known as Fusarium wilt in narrow-leafed lupine. During humid and hot summers, these diseases can decimate white, yellow and narrow-leafed lupine crops, in particular in organic farming systems. The results of our study indicate that the use of jasmonates in early stages of growth could enhance flavone and flavonoid biosynthesis in seedlings and significantly increase their resistance to fungal infections (Ferrer et al., 1990). Similar results were reported by (Król et al., 2015) in tomato seedlings treated with MJ. The influence of jasmonates on the composition of selected phenols and their biosynthesis has been described previously (Yan et al., 2006). Jasmonates were also found to influence the content of 7-O-(6″-Omalonyl) glucosylgenistein and 7-O-glucosylgenistein in etiolated cotyledons of white lupine seedlings incubated in the dark for 48 h (Katagiri et al., 2001). Further research is required in view of a steady increase in the area under lupine (in particular narrow-leafed lupine) cultivation. It should also be noted that the popularity of organic farming systems which contribute to biodiversity and limit the use of conventional fungicides is on the rise in the European Union. 6
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plant foods with increased flavonoid content deliver health benefits for animals with cancer, and b) whether all germinating seeds (such as wheat and garden cress sprouts) respond to MJ in the same way as germinating lupine seeds. These research directions seem promising. At least in some European countries in grocery stores you can buy wheat germ or cress sprouts for health-promoting purposes. In our opinion, the health-oriented use of lupine seedlings with the use of MJ in human nutrition is only a matter of time, the need for several years of laboratory and field experiments. The obtained results confirm the previously assumed thesis that MJ has a beneficial effect on the chemical composition of lupine seedlings considered in terms of nutrition. This is very important at a time when buyers are looking for more and more organic products, in the production of which are subject to very strict prohibitions on the use of plant protection products, including fungicides.
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Acknowledgements This study was partially supported by grant No. 12.610.012-300 from the Ministry of Science and Higher Education References Battilani, P., Gualla, A., Dall’Asta, C., Pellacani, C., Galaverna, G., Giorni, P., Caglieri, A., Tagliaferri, S., Pietri, A., Dossena, A., Spadaro, D., Marchelli, R., Gullino, M.L., Costa, L.G., 2011. Phomopsins: an overview of phytopathological and chemical aspects, toxicity, analysis and occurrence. World Mycotoxin J. 4, 345–359. https://doi.org/ 10.3920/WMJ2011.1302. Białecka, B., Kępczyński, J., 2003. Jasmonides in retching and germination of seeds. Post. Biol. Kom 30, 447–459. Cohen, S., Flescher, E., 2009. Methyl jasmonate: a plant stress hormone as an anti-cancer drug. Phytochemistry 70, 1600–1609. https://doi.org/10.1016/j.phytochem.2009. 06.007. Creelman, R.A., Mullet, J.E., 1997. Biosynthesis and action of jasmonates in plants. Annu. Rev. Plant Physiol. Plant Mol. Biol. 48, 355–381. https://doi.org/10.1146/annurev. arplant.48.1.355. Farmer, E.E., Ryan, C.A., 1990. Interplant communication: airborne methyl jasmonate induces synthesis of proteinase inhibitors in plant leaves. Botany 87, 7713–7716. https://doi.org/10.1073/pnas.87.19.7713. Feldheim, W., 2000. The use of lupins in human nutrition. Lupin, an anc. Crop new millenn. In: Proc. 9th Int. Lupin Conf. Klink/Muritz, Ger. 20-24 June, 1999. pp. 434–437. Ferrer, M.A., Pedreño, M.A., Calderón, A.A., Muñoz, R., Ros Barceló, A., 1990. Distribution of isoflavones in lupin hypocotyls. Possible control of cell wall peroxidase activity involved in lignification. Int. J. Plant Biol. 79, 610–616. https://doi. org/10.1111/j.1399-3054.1990.tb00033.x. Fingrut, O., Flescher, E., 2002. Plant Stress Hormones Suppress the Proliferation and Induce Apoptosis in Human Cancer Cells. https://doi.org/10.1038/sj.leu.2402412. Flescher, E., 2007. Jasmonates in cancer therapy. Cancer Lett. 245, 1–10. https://doi.org/ 10.1016/j.canlet.2006.03.001. Fonseca, S., Chico, J.M., Solano, R., 2009. The jasmonate pathway: the ligand, the receptor and the core signalling module. Curr. Opin. Plant Biol. 12, 539–547. https:// doi.org/10.1016/j.pbi.2009.07.013. Grzesiuk, A., Dębski, H., Horbowicz, M., 2008. Wpływ wybranych czynników na akumulację antocyjanów w roślinach. Postępy Nauk Rol. 60.
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Original Research Article
The influence of exogenous methyl jasmonate on the germination and, content and composition of flavonoids in extracts from seedlings of yellow and narrow-leafed lupine
T
Kazimierz Zalewskia, Bartosz Nitkiewicza,*, Mariusz Stolarskib, Ryszard Amarowiczc, Adam Okorskid a
Department of Biochemistry, Faculty of Biology and Biotechnology, University of Warmia and Mazury, Olsztyn, Poland Department of Plant Breeding and Seed Production, University of Warmia and Mazury, Olsztyn, Poland Division of Food Science, Institute of Animal Reproduction and Food Research of the Polish Academy of Sciences, Olsztyn, Poland d Department of Entomology, Phytopathology and Molecular Diagnostics, University of Warmia and Mazury, Olsztyn, Poland b c
A R T I C LE I N FO
A B S T R A C T
Keywords: Jasmonates JA-Me Flavonoids Lupine seedlings
The aim of this study was to determine the influence of exogenous methyl jasmonate (MJ) on the content and composition of flavonoids (isoflavones) in extracts from hypocotyls (with cotyledons) and radicles of yellow lupine (Lupinus luteus L.) var. Polo and in extracts from radicles of narrow-leafed lupine (Lupinus angustifolius) var. Graf. Lupine seeds were harvested when fully ripe. Two months after harvest, the effect of various MJ concentrations (10−6 M to 10-3M) on seed viability, seed vigor and the content and composition of flavonoids in extracts from seedlings that emerged from germinated lupine seeds (72 h, 20 °C) was determined. At high concentrations (10-4 M to 10-3 M), MJ suppressed the germination rate and germination capacity of seeds and decreased the growth rate of seedlings of the analyzed varieties of yellow and narrow-leafed lupines in the first 5 days of growth. In seedlings, MJ significantly increased the content of isoflavones (including daidzin, genistin, daidzein, and genistein) in 3-day-old hypocotyls (with cotyledons) and radicles of yellow lupine. This correlation was also observed in the hypocotyls (with cotyledons) and radicles of 3-day-old narrow-leafed lupine seedlings treated with MJ. Narrow-leafed lupine seeds were more sensitive to exogenous MJ then yellow lupine seeds during germination.
1. Introduction The structures and biosynthetic pathways of jasmonates, mainly jasmonic acid (JA) and methyl jasmonate (MJ), are similar to those of prostaglandins found in animals. Jasmonates have various effects and are capable of inducing (microtubule decomposition, phytoalexin and alkaloid synthesis, tuber formation, shoot shortening), stimulating (aging, chlorophyll degradation, respiration, and leaf shedding) and inhibiting (Rubisco synthesis, photosynthesis, cell division, embryogenesis, flowering, and seed germination) various biological processes (Sembdner and Parthier, 1993). Exogenous MJ modifies the activity of α-D-galactosidase and the composition of carbohydrates in yellow lupine seedlings (Zalewski et al., 2010). Foliar-applied MJ changes the expression of genes encoding fruit ripening, pollen production, shoot
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growth, root hair growth and resistance to pathogens (Creelman and Mullet, 1997). Jasmonates play an important role in the production of secondary metabolites, including flavonoids. Mechanical injury, pest damage and fungal infections lead to the release of linolenic acid from membrane lipids. Linolenic acid is converted to JA in the octadecanoid pathway. In cells, higher concentrations of JA influence the expression of various genes involved in defense. Jasmonic acid also stimulates anthocyanin biosynthesis (Grzesiuk et al., 2008). Jasmonates resemble abscisic acid in many respects, in particular with regard to plant responses to stress (Parthier, 1991). Jasmonic acid and MJ are the key signaling compounds that induce the expression of genes encoding inhibitors of proteinases (proteins that protect plants against insects) in response to plant damage (Farmer and Ryan, 1990). Methyl jasmonate increases the transcription of genes encoding defensins (PDE 1.2) and
Corresponding author at: Department of Biochemistry, Faculty of Biology and Biotechnology, University of Warmia and Mazury, Oczapowskiego 1A, 10-719, Olsztyn, Poland. E-mail addresses: [email protected] (K. Zalewski), [email protected] (B. Nitkiewicz), [email protected] (M. Stolarski), [email protected] (R. Amarowicz), [email protected] (A. Okorski). https://doi.org/10.1016/j.jfca.2019.103398 Received 16 July 2019; Accepted 27 December 2019 Available online 28 December 2019 0889-1575/ © 2019 Elsevier Inc. All rights reserved.
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Table 1 Viability and vigor of yellow lupine (var. Polo) and narrow-leafed lupine (var. Graf) seeds. Standard deviation is expressed as the number of significant digits. Combination
Germination rate (10 days for yellow lupine and 5 days for narrow-leafed lupine, 20 °C) (%)
Yellow lupine (Lupinus luteus) Control 81.50 ± 1.2 MJ x 10 −6M 81.00 ± 3.1 −5 MJ x 10 M 79.00 ± 3.3 MJ x 10 −4M 64.50 ± 4.0 −3 M 42.25 ± 2.1 MJ x 10 Narrow-leafed lupine (Lupinus angustifolius) Control 75.50 ± 2.4 MJ x 10 −6M 76.00 ± 3.6 MJ x 10 −5M 63.50 ± 3.0 MJ x 10 −4M 52.25 ± 2.1 MJ x 10 −3M 34.50 ± 1.9
Germination capacity (21 days for yellow lupine and 10 days for narrowleafed lupine, 20 °C) (%)
Seedling growth analysis (germination 5 days, 20 °C) Primary root lenght (mm)
Hypocotyl lenght (mm)
Fresh weight of root (g)
Fresh weight of hypocotyl with cotyledons (g)
94.50 ± 2.0 95.00 ± 2.5 91.50 ± 2.5 82.50 ± 2.0 57.00 ± 0.7
42.23 ± 2.5 38.62 ± 0.5 22.70 ± 1.8 15.61 ± 2.1 12.35 ± 0.6
35.2 ± 2.8 34.9 ± 2.9 26.3 ± 1.9 23.5 ± 2.2 12.7 ± 2.3
1.73 ± 0.22 1.67 ± 0.34 1.45 ± 0.20 1.32 ± 0.13 1.13 ± 0.08
5.71 ± 0.26 5.50 ± 0.33 4.36 ± 0.30 3.98 ± 0.24 3.34 ± 0.31
92.25 ± 4.2 93.00 ± 4.3 81.25 ± 4.5 73.50 ± 3.5 42.50 ± 2.9
29.82 ± 2.1 28.33 ± 2.3 22.39 ± 2.0 16.51 ± 1.8 5.10 ± 1.6
34.03 ± 2.8 34.71 ± 2.9 27.43 ± 3.1 15.90 ± 2.5 4.41 ± 1.0
1.07 ± 0.11 1.08 ± 0.10 0.86 ± 0.09 0.56 ± 0.05 0.11 ± 0.03
5.25 ± 0.23 5.17 ± 0.34 4.21 ± 0.41 3.38 ± 0.27 3.13 ± 0.11
Company, St. Paul, USA). Germination rates of yellow and narrowleafed lupine seeds were determined after 10 and 5 days of incubation, respectively (International Seed Testing Associacion (ISTA), 1996). Organic solvents were purchased from Merck (Warsaw, Poland). Trifluoroacetic acid was obtained from Sigma-Aldrich (Poznań, Poland). Standards of daidzin, daidzein, genistin, genistein, and apigenin were purchased from Extrasynthese (Genay Cedex, France).
thionins (THI 2.1), proteins with antibacterial properties (Farmer and Ryan, 1990; Szczegielniak, 2006). Jasmonic acid-dependent signaling pathways activate systemic responses, whereas JA-independent signaling pathways are involved in tissue repair and pathogen defense (Szczegielniak, 2006). According to (Fonseca et al., 2009), jasmonates regulate plant development and adaptation to adverse environmental conditions by deactivating selected natural plant hormones. When applied directly to leaves, JA increases the total phenolic content of basil plants. As an abiotic elicitor, JA could be used to enhance the biological activity and health benefits of sweet basil landraces (Malekpoor et al., 2016). The resistance of tomato seedlings to Fusarium oxysporum f. sp. lycopersici increased 4 weeks after inoculation when tomato seeds had been immersed in a solution of 0.1 mM methyl jasmonate for 1 h. At concentrations of 0.01, 0.1 and 1 mM, MJ inhibited spore germination and the growth of the fungal strain in vitro (Król et al., 2015). The MJ-induced increase in the content of phenolic compounds in plants delivers health benefits to consumers. The positive influence of jasmonates on reducing the development of some human cancer diseases has already been proven (Majewska-Wierzbicka et al., 2012; Yach et al., 2004). Research indicates that jasmonates reduce the risk of certain cancers in humans (Majewska-Wierzbicka et al., 2012). Methyl jasmonate inhibits the proliferation of neuroma and prostate cancer cells, and it significantly decreases the risk of prostate cancer (Tong et al., 2008). Spraying friut with MJ before harvest has positive health implications for consumers (Reyes-Díaz et al., 2016). There is growing evidence to indicate that this plant hormone has health benefits for humans and animals, therefore; it will continue to be a subject of debate in the future (Cohen and Flescher, 2009; Umukoro et al., 2011). Seeds with proteins as their basic material react more strongly than oilseeds eg. sunflower seeds (Białecka and Kępczyński, 2003). This was one of the reasons for using lupine seeds in experiments. In addition, several species of lupins are cultivated in Poland, but only eleven species are grown on the production fields. Three of them are grown for fodder purposes: yellow, narrow-leaved and white lupins. The growing area is about 40,000 ha in 2017 and around 60,000 ha in 2018.
2.1. Germination Lupine seeds were incubated in solutions of 10−3 M, 10-4 M, 10-5 M or 10-6 M MJ (20 °C). Seeds germinated in distilled water were served as the control. After 3 days of germination, hypocotyls with cotyledons and radicles were isolated from yellow lupine seedlings. For comparative purposes, only radicles were isolated from narrow-leafed lupine seedlings, and they were used in flavone and isoflavone analyses. 2.2. Seedling growth analysis Seedling growth was analyzed after 5 days of seed incubation (100 seeds in four replications) in distilled water in the dark at a temperature of 20 °C, different concentrations of the applied growth regulator. The following seedling parameters were analyzed: radicle length, hypocotyl length, fresh weight of hypocotyls with cotyledons, and fresh radicle weight. 2.3. Extraction of phenolic compounds Isolated hypocotyls with cotyledons and isolated radicles were frozen in liquid nitrogen and ground in an impact mill. Phenolic compounds were extracted in three replications of 15 min each, with 70% methanol, at 40 °C, in a Julabo SW-22 shaking water bath (WITKO, Warsaw, Poland). Methanol extracts were combined, and organic solvent was evaporated in a Büchi R-200 vacuum evaporator (Büchi, Warsaw, Poland) at 40 °C. The remaining water was removed by freeze drying (Labconco, Kansas City, MO, USA).
2. Materials and methods 2.4. HPLC analysis The study was carried out on yellow and narrow-leafed lupine seedlings. Seeds were harvested when fully ripe; they were then dried and stored in linen bags at 16−18 °C and a mean humidity of 60–65%. The average thousand seed weight (at 11% moisture content) was determined to be 105.2 g for yellow lupine and 143.4 g for narrow-leafed lupine. Germination capacity was determined as the percentage of yellow lupine seeds that germinated after 21 days of imbibition and the percentage of narrow-leafed lupine seeds that germinated after 10 days of imbibition on moistened germination paper (Anchor Paper
The obtained extract was dissolved in 70% methanol and passed through a 0.45 μm filter. The HPLC analysis was carried out in a Shimadzu HPLC system (Shimadzu, Kyoto, Japan) composed of two LC10AD pumps, an SPD-M 10A photodiode detector and an SCTL-10A controller. Phenolic compounds were separated on a Luna C18 column (4 x 250 mm, 5 μm; Phenomenex, Torrance, CA, USA) in a 5–40% aqueous solution of acetonitrile with the addition of 0.1% trifluoroacetic acid (TFA) for 50 min. The injection volume was 20 μl, the 2
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Fig. 1. Chromatographic separation (HPLC) of isoflavones and flavones isolated from the radicles of narrow-leafed lupine seedlings. A – control; B –10−4M MJ; C-10M MJ.
3
flow rate was 1 ml/min, and detection wavelength was 260 nm and 320 nm. For quantitative analysis four standards of isoflavones (daidzein, daidzin, genistein, genistin) and one standard of flavone (apigenin) were used. The external standard method for quantitative analysis. The linearity of calibration curves (standard peak area versus standard concentration) was characterized by correlation coefficients r > 0.995. Isoflavones were determined at 260 nm and flavones at 320 nm. The content of flavones was expressed as apigenin equivalents. All the chemical determinations were performed in triplicate. The results are reported as means ± SD (Table 1). Differences in the content of each isoflavones were statistically evaluated by ANOVA and Tukey’s posttest comparisons. Statistical calculations were performed using:
STATISTICA software ver. 10 (StatSoft, Inc.). 3. Results An analysis of lupine seed viability demonstrated that germination capacity exceeded 90% in both lupine species (control – germination in H2O). High concentrations of MJ (10−3 M, 10-4 M and partially 10-5 M) decreased germination rate and germination capacity. Narrow-leafed lupine seeds were more sensitive to exogenous MJ then yellow lupine seeds. An analysis of 5-day-old seedlings revealed similar results (Table 1). Methyl jasmonate exerted a more pronounced effect on yellow lupine radicles (smaller increase in length and lower fresh weight of radicles of hypocotyls). 3
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Fig. 2. UV–vis absorption spectra of isoflavones from lupine seedling extracts and the genistein standard. Numbers 1–4 correspond to the peaks in Fig. 1.
concentrations of 10−6 M to 10-5 M, and c) the highest concentrations of MJ applied during seed germination (10-4 M and, 10-3 M). All differences between the above groups were statistically significant. The content of compounds 1–4 was expressed in terms of the apigenin equivalent, and the content of compounds 7 and 8, – in terms of the genistein equivalent. For the control sample (germination in distilled water) the content of compounds 1–4 was expressed in terms of the genistin equivalent, and the content of compounds 5–8, – in terms of the apigenin equivalent. The extracts from the radicles of germinating narrow-leafed lupine seeds (control sample) contained 7 flavonoids, including 17.94 mg of isoflavones/g of the extract and 5.31 mg of flavones/g of the extract (Fig. 5). The presence of jasmonate in incubation mixtures induced changes in the flavonoid content of roots. In samples exposed to MJ at a concentration of 10−4 M, the flavonoid content of roots increased (genistin and compound no. 4) or decreased (compound no. 1, daidzin, daidzein and compound no. 8). Exposure to the highest jasmonate concentration significantly increased the content of the analyzed compounds relative to the control sample (Fig. 5), in particular compound 1 of the genistin family the content of which increased more than 5-fold. Daidzin and compound 4 were the predominant components in the group of 8 flavonoids identified in hypocotyls with cotyledons isolated from the radicles of yellow lupine seedlings (Table 2). In the control sample compound 7 (Table 2) predominated in the group of flavones. Only the content of genistin derivative (compound no. 1 in Table 2) did
The HPLC chromatograms of the analyzed extracts of phenolic compounds isolated from the radicles of narrow-leafed lupine seedlings (Fig. 1) revealed four main isoflavone peaks (1–4) registered at 260 nm (retention times: 23.8, 28.4, 29.5, and 33.9 min) and four flavone peaks (5–8) registered a 320 nm (retention times: 30.8, 31.5, 35.3, and 44.7 min) (Fig. 1). It should be noted that peak 6 was visible only in the chromatogram of the sample incubated with the highest concentration of MJ. The spectra of compounds corresponding to peaks 1–4 were characterized by the maximum absorption at 259–260 nm. The spectra of those compounds were identical to the genistein spectrum (Fig. 2). Two absorption bands were observed in the spectra of compounds corresponding to peaks 5–8. The maximum absorption was noted at 267 nm for the first band and at 324−336 nm for the second band. The spectra of those compounds were highly similar to the apigenin spectrum (Fig. 3). Seven flavonoids were identified in the radicles of germinating yellow lupine seeds, 4 of which were detected based on the simultaneous separation of the reference standards. The data presented in Fig. 4 indicate that the content of the analyzed compounds varied from 0.71 μg to more than 8 μg per mg under control conditions. All concentrations of exogenous MJ enhanced the biosynthesis of all flavonoids, and the synthesis of an apigenin group compound (no. 7 in Fig. 4) increased 14-fold. The results presented in Fig. 4 can be divided into three significantly different groups: a) control, b) MJ 4
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Fig. 3. UV–vis absorption spectra of flavones from lupine seedling extracts and the apigenin standard. Numbers 5–8 correspond to the peaks in Fig. 1. Fig. 4. Flavonoid content of methanol extracts from the radicles of germinating yellow lupine seeds (μg/mg of extract). Compounds: 1,4,7 expressed as apigenin equivalents; 2,5,6 expressed as genistin equivalents (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).
4. Discussion
not differ significantly between the experimental treatments. The content of the remaining flavonoids differed significantly at all concentrations of MJ relative to the control sample. The greatest increase was observed in daidzein content (more than 12-fold) in response to the highest concentration of MJ during seed incubation.
MJ causes less water accumulation in germinated seeds (cell wall lignification), limiting the growth of seedlings. Therefore, the content of flavonoids is presented as μg per mg of methanol extract obtained, which is common in many publications. The observed increase in the flavonoid content of lupine seedlings
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Fig. 5. The content of individual phenolic compounds in extracts from narrow-leafed lupine radicles (μg/mg of extract; the quantity of extracts was identical in all samples). Compounds: 1,4,7,8 expressed as apigenin equivalents; 2,5,6 expressed as genistin equivalents. Table 2 The flavonoid content of methanol extracts from the hypocotyls (with cotyledons) of germinating yellow lupine seeds (μg/mg of extract). Compounds 1,4,7,8 expressed as apigenin equivalents. Flavonoid derivative
Control
MJ 10−6 M
MJ 10−5 M
MJ 10−4 M
MJ 10−3 M
1 Daidzin Genistin 4 Daidzein Genistein 7 8 Together
1.75 ± 0.09 3.65 ± 0.02 2.41 ± 0.11 3.65 ± 0.13 0.26 ± 0.01 1.23 ± 0.07 4.36 ± 0.24 0.55 ± 0.03 17.98 ± 0.63
2.12 ± 0.11 6.16 ± 0.34 3.92 ± 0.18 6.85 ± 0.24 0.21 ± 0.01 2.25 ± 0.14 6.35 ± 0.35 0.72 ± 0.04 28.58 ± 1.26
1.97 ± 0.10 4.12 ± 0.23 4.06 ± 0.18 4.01 ± 0.14 0.89 ± 0.05 1.98 ± 0.12 5.10 ± 0.28 1.10 ± 0.06 23.23 ± 1.19
2.26 ± 0.11 9.08 ± 0.50 3.85 ± 0.17 10.35 ± 0.36 2.89 ± 0.15 3.14)5 ± 0.19 8.02 ± 0.45 0.83 ± 0.05 40.43 ± 2.23
1.85 ± 0.09 7.03 ± 0.39 3.75 ± 0.17 8.69 ± 0.60 3.20 ± 0.17 3.11 ± 0.19 8.36 ± 0.46 2.36 ± 0.13 38.35 ± 2.28
The mycotoxin content of seeds of fungi infested plants is another important consideration. Lupine seeds are processed into lupine flour, meat substitutes, plant milk, yogurt and cheese (Feldheim, 2000; Hill, 1986). Legume seeds are also widely used in the production of animal feeds. Members of the genus Fusarium are among the most pathogenic toxin-producing fungi that colonize lupine plants (Selwet, 2010). Not all seeds of fungi-infected plants contain mycotoxins. However, the use of contaminated seeds in food and feed production poses significant health risks (Zain, 2011). Mycotoxins are not degraded at temperatures below 250 °C and are not eliminated during cooking. Therefore, further research into effective methods of eliminating mycotoxins from foodstuffs and feedstuffs, including jasmonate treatments of seeds (pre-imbibition) and seedlings, is required, and its results should have direct practical applications. Flavonoids are a diverse group of phenolic compounds that occur naturally in plants. These secondary metabolites deliver health benefits (Cohen and Flescher, 2009; Ranilla et al., 2009; Reyes-Díaz et al., 2016). Apigenin is a naturally occurring flavonoid in parsley, thyme, chamomile and red pepper, and the biosynthesis of its derivatives in lupine seedlings is stimulated by exogenous MJ. Apigenin speeds up the formation of nerve cells from stem cells, and strengthens connections between brain cells, which can improve memory and learning (Paulsen et al., 2011). For these reasons, apigenin may have the potential to treat Parkinson’s disease and Alzheimer’s disease (Miller et al., 2008). Jasmonates have also been found to suppress cell proliferation and induce apoptosis in human and mouse breast cancer, prostate cancer, melanoma, lymphoblastic leukemia (Fingrut and Flescher, 2002) and lymphoma cell lines (Reischer et al., 2007). The anticarcinogenic effects of jasmonates have been described in a review article (Flescher, 2007). Further research is required to address two key issues: a) whether
treated with exogenous MJ should be analyzed from several points of view because research into flavonoids pursues several objectives. One of them is the influence of flavonoids on plant development and resistance to pathogens. Other lines of research investigate the health benefits of flavonoids and other phenols in plant-based foods (Ranilla et al., 2009). Attempts have also been made, in particular in Australia and New Zealand, to determine mycotoxin levels in lupine seeds which are used in the production of animal feeds and can pose a significant health threat, especially to sheep and pigs (Battilani et al., 2011). The current study, which investigated the influence of exogenous MJ on germinating lupine seeds, falls within the above mentioned research scope. Lupines are particularly susceptible to fungal infections which cause anthracnose and Fusarium diseases, known as Fusarium wilt in narrow-leafed lupine. During humid and hot summers, these diseases can decimate white, yellow and narrow-leafed lupine crops, in particular in organic farming systems. The results of our study indicate that the use of jasmonates in early stages of growth could enhance flavone and flavonoid biosynthesis in seedlings and significantly increase their resistance to fungal infections (Ferrer et al., 1990). Similar results were reported by (Król et al., 2015) in tomato seedlings treated with MJ. The influence of jasmonates on the composition of selected phenols and their biosynthesis has been described previously (Yan et al., 2006). Jasmonates were also found to influence the content of 7-O-(6″-Omalonyl) glucosylgenistein and 7-O-glucosylgenistein in etiolated cotyledons of white lupine seedlings incubated in the dark for 48 h (Katagiri et al., 2001). Further research is required in view of a steady increase in the area under lupine (in particular narrow-leafed lupine) cultivation. It should also be noted that the popularity of organic farming systems which contribute to biodiversity and limit the use of conventional fungicides is on the rise in the European Union. 6
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plant foods with increased flavonoid content deliver health benefits for animals with cancer, and b) whether all germinating seeds (such as wheat and garden cress sprouts) respond to MJ in the same way as germinating lupine seeds. These research directions seem promising. At least in some European countries in grocery stores you can buy wheat germ or cress sprouts for health-promoting purposes. In our opinion, the health-oriented use of lupine seedlings with the use of MJ in human nutrition is only a matter of time, the need for several years of laboratory and field experiments. The obtained results confirm the previously assumed thesis that MJ has a beneficial effect on the chemical composition of lupine seedlings considered in terms of nutrition. This is very important at a time when buyers are looking for more and more organic products, in the production of which are subject to very strict prohibitions on the use of plant protection products, including fungicides.
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