Cutting Management Approaches to Understand Phytoestrogens Accumulation in Forage Legume Species Used in Dairy Production Systems

Cutting frequency and cutting height study

Research results

The total phytoestrogen concentration in red clover varied based on cutting frequency (3X vs 5X) but not cutting height (ANOVA, frequency (F): p<0.01, height (H): p=0.69). Total phytoestrogen concentration in red clover was higher in 5X than in 3X cutting (Fig. 2A). In white clover, total phytoestrogen concentration was not influenced by either cutting frequency or cutting height (ANOVA, F: p=0.06; H: p=0.17, Fig. 2B). Figure 2. Bar chart showing effects of cutting frequency on total phytoestrogen concentration in red clover (A) and white clover (B). Whiskers indicate ±1 SE of the mean (n = 24 for 3X and 40 for 5X). Treatments sharing the same letter are not significantly different (α = 0.05) as determined by Tukey’s HSD test. NS indicates no significant differences.

The concentration of total phytoestrogens in red clover was approximately 73 times higher compared to white clover. Formononetin and biochanin A were the most abundant phytoestrogens in both species, accounting for 85% (red clover) and 65% (white clover) of the total (Fig. 3). In red clover, formononetin alone accounted for nearly half of the total phytoestrogen content. Prunetin concentration was low in both clover species, while coumestrol was only reported in white clover and made up just 4% of the total phytoestrogen content.Figure 3. Pie chart showing the percentage of individual phytoestrogen compounds in red clover (A) and white clover (B).

Concentrations of individual phytoestrogen compounds in red clover showed contrasting responses to cutting frequency. Formononetin concentration was 56% higher in 5X compared to 3X (ANOVA, F: p<0.01, Fig. 4A). In contrast, concentrations of daidzein (ANOVA, F: p<0.01, Fig. 4D) and glycitein (ANOVA, F: p<0.05, Fig. 4F) were 19.8% and 19.4% higher, respectively in 3X compared to 5X. Cutting frequency did not influence the concentration of biochanin A, genistein, or prunetin in red clover (ANOVA, F: p>0.05, Fig. 4B, C, and E). Concentrations of individual phytoestrogen compounds in red clover did not differ by cutting height (ANOVA, H: p>0.05) and there were no interactions between cutting frequency and height (ANOVA, F×H: p>0.05). There was no effect of cutting frequency and cutting height on individual phytoestrogen concentrations in white clover (ANOVA, F: p>0.05, H: p>0.05, data not shown).Figure 4. Bar graph showing effect of cutting frequency on individual phytoestrogen concentration in red clover; formononetin (A), biochanin A (B), prunetin (C), daidzein (D), genistein (E), and glycitein (F). Whiskers indicate ±1 SE of the mean (n = 24 for 3X and 40 for 5X). Treatments sharing the same letter are not significantly different (α = 0.05) as determined by Tukey’s HSD test. NS indicates no significant differences.

Phytoestrogen concentration over the harvest season

In both red clover and white clover, total phytoestrogen concentrations in the 5X treatment varied dramatically over the growing season. In red clover, the total phytoestrogen concentration increased by 89.7% in second cutting compared to the first cutting. The highest concentrations of total phytoestrogens in red clover were observed in the second and third cuttings (ANOVA, cutting time: p<0.01, Fig. 5A).

Figure 5. Line graph showing effects of cutting time on total phytoestrogen concentration in red clover (A) and white clover (B) in the 5X treatment. Whiskers indicate ±1 SE of the mean (n = 8). Treatments sharing the same letter are not significantly different (α = 0.05) as determined by Tukey’s HSD test.    

A somewhat similar pattern was observed in white (ANOVA, cutting time: p<0.01, Fig. 5B). Total phytoestrogen concentration at the third cutting time was more than eight times higher than the first cutting and nearly three times higher compared to the second cutting but dropped dramatically in the fourth and fifth cuttings (Fig. 5B). Total phytoestrogen concentrations did not differ among cutting times in the 3X treatment in either red clover or white clover (ANOVA, cutting time: p>0.05, data not shown).

Discussion

These results reveal large differences in phytoestrogen concentrations between two commonly grown clover species. Phytoestrogen concentrations were 73 times higher in red clover compared to white clover and this result is in line with previous studies [26,32]. The total phytoestrogen concentration (5.01 mg/g) and relative proportion of different individual phytoestrogen compounds in red clover are comparable with those observed in a previous greenhouse study conducted on the same cultivar [33]. Formononetin and biochanin A are two most abundant phytoestrogens in both clover species, and this finding is consistent with other studies [32,34,35]. We observed 34.6% higher total phytoestrogen concentration in red clover cut more frequently (5X) compared to when it was cut less frequently (3X). After each cutting, plants regrow their leaves to restore their photosynthetic area. In the 5X treatment, aboveground biomass was cut more frequently, and plants had less time to recover, which likely altered the physiological activity of plants by interrupting their energy supply and triggering the re-distribution of assimilates and mobilization of carbohydrate reserves [36]. Thus, the frequent cutting likely results in a stress response in red clover that may trigger upregulation of phytoestrogen production. In the case of white clover, total phytoestrogen concentration was not influenced by cutting frequency. Variation between the two clover species in their phytoestrogen response to cutting frequency could be due to variation in the capacity to utilize root reserves as well as their growth pattern [37,38]. White clover grows laterally and is shorter compared to red clover, and so it may be less stressed by cutting than is red clover because more leaf area remains undamaged [39]. Previous studies have reported that isoflavone concentrations in red clover can increase soon after defoliation [40,41]. After a cutting event, both species rely on their remaining leaf area and root reserves to recover [42,43]; however, in red clover less leaf area and stem material remains undamaged compared to white clover. Interestingly, however, we did not find an effect of cutting height on phytoestrogen accumulation in either species. It is possible that the difference in cutting height between our treatments (5cm vs 10cm residual height) was not great enough to result in substantive differences residual leaf area or root reserves, and therefore a stress response in terms of their production of phytoestrogens. Unfortunately, we did not measure residual leaf area (or root reserves) after any of the harvests.

We observed striking differences in phytoestrogen concentrations at different cutting times in the 5X treatment. The variation in isoflavone concentration at different cutting times over the growing season may have been associated with variation in the weather [44]. In our previous research on red clover, we found that drought stress more than doubled the phytoestrogen levels in red clover [33]. During the second and third cutting times, air temperatures were relatively high, and there was relatively low precipitation, which could have led to short-term drought conditions that increased the phytoestrogen levels in the plants. It is also possible that concentrations of some phytoestrogen compounds are naturally dynamic. In previous research on red clover, genistein and daidzein content were observed to increase from June to July, while biochanin A and formononetin concentrations peaked in early September [45].

Elevated temperature and CO₂study

Phytoestrogen concentrations

Effects of the temperature and CO₂ treatments were visually apparent in both legume species (Figure 6). Phytoestrogen concentrations in cowpea were below the detection level regardless of treatment (data not shown). In contrast, phytoestrogen concentrations in red clover were well above the detection level and influenced by treatment (ANOVA, treatment: p<0.01; Figure 7). Compared to red clover grown under the ambient control conditions, elevated temperature (eT), both when applied alone and in combination with elevated CO₂ (eCO₂), resulted in a 50% and 53% reduction in total phytoestrogen concentration, respectively. Total phytoestrogen concentration in red clover did not differ compared to the ambient control when eCO₂ was applied alone.Figure 6. Representative photographs of cowpea (upper row) and red clover (lower row) in each temperature and CO₂ treatment following 10 days of exposure.

Figure 7. Bar graph showing total phytoestrogen concentration in red clover (mg/g DM) after 10 days of exposure to eT and eCO₂. Bars indicate standard errors of the mean (n = 4). Treatments sharing the same letter are not significantly different (α = 0.05) as determined by Tukey’s HSD test.

In red clover, individual phytoestrogen compounds varied in their responses to the temperature and CO₂ treatments (Figure 8). Compared to the ambient control, concentrations of formononetin were 49% and 47% lower, respectively, in the eT and eT+eCO₂ conditions (ANOVA, treatment: p<0.05, Figure 8a). Similarly, biochanin A concentrations were 59% lower in the eT and 63% lower in eT+eCO₂ compared to the control (ANOVA, treatment: p<0.05, Figure 8b). The concentration of daidzein was reduced by 43% in eCO₂ and 42% in eT+eCO₂ condition (ANOVA, treatment: p<0.01, Figure 8c). Genistein, glycitein and prunetin concentrations were not influenced by the treatments.

Figure 8. Effects of exposure to eT and eCO₂ on concentrations (mg/g DM) of individual isoflavones formononetin (a), biochanin A (b), daidzein (c), genistein (d), glycitein (e), and prunetin (f) in red clover. Bars indicate standard errors of the mean (n = 4). Treatments sharing the same letter are not significantly different (α = 0.05) as determined by Tukey’s HSD test.

Discussion

To the best of our knowledge, ours is the first study to quantify phytoestrogen concentration in the vegetative component of cowpea. While previous research has demonstrated that cowpea seeds contain low concentrations of phytoestrogens [46,47], and that concurrent application of high temperatures and increased levels of CO₂ can decrease isoflavone concentrations in seeds of other legumes such as soybean [48], we found phytoestrogen concentrations in cowpea vegetative tissues to be below the level of detection. Whether cowpea might upregulate phytoestrogen concentrations in its vegetative tissues under other types of environmental stress, such as drought or pest damage, or how eT or eCO₂ may influence phytoestrogen concentrations in cowpea seeds, remains unknown.

In contrast to cowpea, red clover had relatively high total concentrations of phytoestrogens (3.9 mg/g in the ambient control) and these concentrations were sensitive to eT. Compared to the control treatment, eT alone and in combination with eCO₂ reduced total phytoestrogen concentration by 50% (2.0 mg/g) and 53% (1.8 mg/g), respectively. Much of this total was made up of formononetin and biochanin A, which together accounted for 83-96% of the total phytoestrogen measured, depending on treatment, which is in line with previous studies conducted on red clover [33,44]. Baseline formononetin concentrations in red clover can vary depending on a range of factors, including cultivar, plant maturity, and growing conditions [49,50]. Formononetin concentrations we observed in red clover grown under our ambient control treatment conditions were lower than those observed in a previous greenhouse study conducted on the same cultivar at similar maturity (3.2 mg/g), possibly due to differences in the ambient growing conditions in the climate-controlled growth chamber compared to those in a greenhouse [33,44].

Our finding that eT depressed total phytoestrogen production in red clover vegetative tissues is congruent with previous research examining the effects of temperature on phytoestrogen contents of soybean seeds and pods [48,51,52]. The decrease in isoflavone synthesis in some legumes under heat stress may occur as plants prioritize resources to synthesize heat shock proteins and to activate other protective mechanisms at the expense of isoflavone synthesis [53]. Determining whether this mechanism explains our observations in red clover requires further research. Isoflavone synthesis through the phenylpropanoid pathway requires carbon, with about 20% of photosynthetic carbon going toward making phenolic compounds, including flavonoids [54,55]. In soybean, increased levels of CO₂ has been shown to increase isoflavone concentrations in seeds and can offset the declines in concentrations due to high temperature [48,52]. We observed that red clover exposed to eCO₂ either alone or in combination with eT did not appreciably increase formononetin and biochanin A concentrations, suggesting that synthesis of these compounds was not carbon limited.

Objective 3: Disseminate phytoestrogen data to stakeholders.

We have prepared two manuscripts from this project for submission to peer-reviewed journals. One manuscript, which we plan to submit to the journal Crop Science, reports on the effects of cutting frequency and height phytoestrogen levels in red clover and white clover. The other manuscript, which reports the effects of elevated temperature and CO₂ on phytoestrogens in red clover and cowpea, will be submitted to Scientific Reports. We are also working with UNH Extension to publish an extension article reporting the phytoestrogen concentrations of the 12 forage legume cultivars grown in our common garden plots (Table 1).

Table 1: Concentrations of phytoestrogen compounds (mg/g DM) and total phytoestrogens measured in different forage legume species and cultivars grown for two seasons in Madbury, NH. Concentrations were measured each August in 2022 and 2023. The values are means (1 standard deviation), n = 4. 

Data are based on two replicates sampled over two years (n=4), except ², 2-two replicates and ³, 3-three replicates; * grown in greenhouse conditions; Phytoestrogen compounds are Formn.-Formononetin, BioA.-Biochanin A, Daid.-Daidzein, Gen.-Genistein, Gly.-Glycitein, Prn.-Prunetin, Kaemp.-Kaempferol, and TotalP.-Total Phytoestrogens.

References

1. Greene, C.; McBride, W. Consumer Demand for Organic Milk Continues to Expand—Can the US Dairy Sector Catch Up? Choices 2015, 30, 1–6.
2. Pereira, A.B.D.; Brito, A.F.; Townson, L.L.; Townson, D.H. Assessing the Research and Education Needs of the Organic Dairy Industry in the Northeastern United States. J. Dairy Sci. 2013, 96, 7340–7348.
3. Li, X.; Peterson, H.H.; Xia, T. Demand for Organic Fluid Milk across Marketing Channels. Agric. Resour. Econ. Rev. 2018, 47, 505–532.
4. Paulson, J.; Jung, H.; Raeth-Knight, M.; Linn, J. Grass vs. Legume Forages for Dairy Cattle. In Proceedings of the Proceedings of the 69^th Annual Minnesota Nutrition Conference, Owatonna, MN. 16–17 Sept.; University of Minnesota. Minnesota Extension Service: Owatonna, MN, 2008; pp. 119–133.
5. Kuhnen, S.; Stibuski, R.B.; Honorato, L.A. Farm Management in Organic and Conventional Dairy Production Systems Based on Pasture in Southern Brazil and Its Consequences on Production and Milk Quality. Animals 2015, 5, 479–494.
6. Steinshamn, H. Effect of Forage Legumes on Feed Intake, Milk Production and Milk Quality–a Review. Anim. Sci. Pap. reports 2010, 28, 195–206.
7. Brito, A.F.; Lange, M.J.; Lange, M.J. The Key Role of Forage Legumes in Organic Dairy Diets: Effects on Your Bottom Line Available online: https://nodpa.com/n/945/The-Key-Role-of-Forage-Legumes-in-Organic-Dairy-Diets-Effects-on-Your-Bottom-Line.
8. Watson, C.A.; Atkinson, D.; Gosling, P.; Jackson, L.R.; Rayns, F.W. Managing Soil Fertility in Organic Farming Systems. Soil use Manag. 2002, 18, 239–247.
9. Carlsson, G.; Huss-Danell, K. Nitrogen Fixation in Perennial Forage Legumes in the Field. Plant Soil 2003, 253, 353–372.
10. Sarelli, L.; Tuori, M.; Saastamoinen, I.; Syrjälä-Qvist, L.; Saloniemi, H. Phytoestrogen Content of Birdsfoot Trefoil and Red Clover: Effects of Growth Stage and Ensiling Method. Acta Agric. Scand. - Sect. A Anim. Sci. 2003, 53, 58–63.
11. Woclawek-Potocka, I.; Piskula, M.K.; Bah, M.M.; Siemieniuch, M.J.; Korzekwa, A.; Brzezicka, E.; Skarzynski, D.J. Concentrations of Isoflavones and Their Metabolites in the Blood of Pregnant and Non-Pregnant Heifers Fed Soy Bean. J. Reprod. Dev. 2008, 54, 807300086.
12. Tucak, M.; Čupić, T.; Horvat, D.; Popović, S.; Krizmanić, G.; Ravlić, M. Variation of Phytoestrogen Content and Major Agronomic Traits in Alfalfa (Medicago Sativa L.) Populations. Agronomy 2020, 10, 87.
13. Hashem, N.M.; Soltan, Y.A. Impacts of Phytoestrogens on Livestock Production: A Review. Egypt. J. Nutr. Feed. 2016, 19, 81–89.
14. Retana-Márquez, S.; Hernández, H.; Flores, J.A.; Muñoz-Gutiérrez, M.; Duarte, G.; Vielma, J.; Fitz-RodrÃguez, G.; Fernández, I.G.; Keller, M.; Delgadillo, J.A. Effects of Phytoestrogens on Mammalian Reproductive Physiology. Trop. Subtrop. Agroecosystems 2011, 15, S129–S145.
15. Tucker, H.A.; Knowlton, K.F.; Meyer, M.T.; Khunjar, W.O.; Love, N.G. Effect of Diet on Fecal and Urinary Estrogenic Activity. J. Dairy Sci. 2010, 93, 2088–2094.
16. Gierus, M.; Koch, M.; Schulz, H. Phytoestrogen Carryover into Cow’s Milk from Legumes-an Overview along the Food Chain. Berichte über Landwirtschaft 2012, 90, 354–379.
17. De Rijke, E.; Aardenburg, L.; Van Dijk, J.; Ariese, F.; Ernst, W.H.O.; Gooijer, C.; Brinkman, U.A.T. Changed Isoflavone Levels in Red Clover (Trifolium Pratense L.) Leaves with Disturbed Root Nodulation in Response to Waterlogging. J. Chem. Ecol. 2005, 31, 1285–1298.
18. Gerard, P.J.; Crush, J.R.; Hackell, D.L. Interaction between Sitona Lepidus and Red Clover Lines Selected for Formononetin Content. Ann. Appl. Biol. 2005, 147, 173–181.
19. Carlsen, S.C.K.; Understrup, A.; Fomsgaard, I.S.; Mortensen, A.G.; Ravnskov, S. Flavonoids in Roots of White Clover: Interaction of Arbuscular Mycorrhizal Fungi and a Pathogenic Fungus. Plant Soil 2008, 302, 33–43.
20. Strauss, L.; Santti, R.; Saarinen, N.; Streng, T.; Joshi, S.; Mäkelä, S. Dietary Phytoestrogens and Their Role in Hormonally Dependent Disease. Toxicol. Lett. 1998, 102, 349–354.
21. Reed, K.F.M. Fertility of Herbivores Consuming Phytoestrogen-Containing Medicago and Trifolium Species. Agriculture 2016, 6, 35.
22. Hoeck, J.A.; Fehr, W.R.; Murphy, P.A.; Welke, G.A. Influence of Genotype and Environment on Isoflavone Contents of Soybean. Crop Sci. 2000, 40, 48–51.
23. Tsao, R.; Papadopoulos, Y.; Yang, R.; Chris Young, J.; Mcrae, K. Isoflavone Profiles of Red Clovers and Their Distribution in Different Parts Harvested at Different Growing Stages. J. Agric. Food Chem. 2006, 54, 5797–5805.
24. Saviranta, N.M.M.; Anttonen, M.J.; Von Wright, A.; Karjalainen, R.O. Red Clover (Trifolium Pratense L.) Isoflavones: Determination of Concentrations by Plant Stage, Flower Colour, Plant Part and Cultivar. J. Sci. Food Agric. 2008, 88, 125–132.
25. Butkutė, B.; Lemežienė, N.; Dabkevičienė, G.; Jakštas, V.; Vilčinskas, E.; Janulis, V. Source of Variation of Isoflavone Concentrations in Perennial Clover Species. Pharmacogn. Mag. 2014, 10, S181–S188.
26. Saloniemi, H.; Kallela, K.; Saastamoinen, I. Study of the Phytoestrogen Content of Goat’s Rue (Galega Orientalis), Alfalfa (Medicago Sutiva) and White Clover (Trifolium Repens). Agric. Food Sci. 1993, 2, 517–524.
27. Höjer, A.; Adler, S.; Purup, S.; Hansen-Møller, J.; Martinsson, K.; Steinshamn, H.; Gustavsson, A.-M. Effects of Feeding Dairy Cows Different Legume-Grass Silages on Milk Phytoestrogen Concentration. J. Dairy Sci. 2012, 95, 4526–4540.
28. Gierus, M.; Kleen, J.; Loges, R.; Taube, F. Forage Legume Species Determine the Nutritional Quality of Binary Mixtures with Perennial Ryegrass in the First Production Year. Anim. Feed Sci. Technol. 2012, 172, 150–161.
29. Sanderson, M.A.; Stair, D.W.; Hussey, M.A. Of Perennial Forages to Stress. Adv. Agron. 1997, 59, 171.
30. Rossiter, R.C. Physiological and Ecological Studies on the Oestrogenic Isoflavones in Subterranean Clover (T. Subterraneum L.). VI. Effects of Defoliation Including Grazing. Aust. J. Agric. Res. 1969, 20, 25–35.
31. Payette, M.; Lima, M.R.M.; Coleman, W.M.; Ashraf-Khorassani, M. Separation Optimization and Quantitative Analysis of Phytoestrogens Employing Reverse-Phase High-Performance Liquid Chromatography with UV-VIS Detection. J. Liq. Chromatogr. Relat. Technol. 2021, 44, 888–896.
32. Andersen, C.; Weisbjerg, M.R.; Hansen-Mller, J.; Sejrsen, K. Effect of Forage on the Content of Phyto-Oestrogens in Bovine Milk. Animal 2009, 3, 617–622.
33. Mandal, P.; Mortensen, D.A.; Brito, A.F.; Wallingford, A.K.; Lima, M.R.M.; Warren, N.D.; Smith, R.G. Water Stress Influences Phytoestrogen Levels in Red Clover (Trifolium Pratense) but Not Kura Clover (T. Ambiguum). J. Agric. Food Chem. 2024, 72, 10247–10256.
34. Mustonen, E.; Tuori, M.; Kurki, P.; Isolahti, M.; Taponen, J.; Vanhatalo, A. Variety, Time of Harvest and Conditions during Growing Season Have Impact on Red Clover Isoflavone Content. Agric. Food Sci. 2018, 27, 102–109.
35. Tucak, M.; Popović, S.; Horvat, D.; Čupić, T.; Krizmanić, G.; Viljevac Vuletić, M.; Ravlić, M. The Characterization of Isoflavone Content in the Croatian Red Clover Collection. Poljoprivreda 2019, 25, 3–11.
36. Briske, D.D.; Richards, J.H. Physiology of Plants Recovering from Defoliation. In Proceedings of the Proceedings of the XVII international grassland congress; 1993; p. 85.
37. Li, R.; Volenec, J.J.; Joern, B.C.; Cunningham, S.M. Seasonal Changes in Nonstructural Carbohydrates, Protein, and Macronutrients in Roots of Alfalfa, Red Clover, Sweetclover, and Birdsfoot Trefoil. Crop Sci. 1996, 36, 617–623.
38. Moot, D.; Black, A.; Lyons, E.M.; Egan, L.M.; Hofmann, R.W. Pasture Resilience Reflects Differences in Root and Shoot Responses to Defoliation, and Water and Nitrogen Deficits.; New Zealand Grassland Association, 2021.
39. Patterson, J.D.; Laidlaw, A.S.; McBride, J. The Influence of Autumn Management and Companion Grass on the Development of White Clover over Winter in Mixed Swards. Grass Forage Sci. 1995, 50, 345–352.
40. McMurray, C.H.; Laidlaw, A.S.; McElroy, M. The Effect of Plant Development and Environment on Formononetin Concentration in Red Clover (Trifolium Pratense L.). J. Sci. Food Agric. 1986, 37, 333–340.
41. Mustonen, E.A.; Tuori, M.; Saastamoinen, I.; Taponen, J.; Wähälä, K.; Saloniemi, H.; Vanhatalo, A. Equol in Milk of Dairy Cows Is Derived from Forage Legumes Such as Red Clover. Br. J. Nutr. 2009, 102, 1552–1556.
42. Meuriot, F.; Avice, J.-C.; Simon, J.-C.; Laine, P.; Decau, M.-L.; Ourry, A. Influence of Initial Organic N Reserves and Residual Leaf Area on Growth, N Uptake, N Partitioning and N Storage in Alfalfa (Medicago Sativa) during Post-Cutting Regrowth. Ann. Bot. 2004, 94, 311–321.
43. Teixeira, E.I.; Moot, D.J.; Brown, H.E.; Pollock, K.M. How Does Defoliation Management Impact on Yield, Canopy Forming Processes and Light Interception of Lucerne (Medicago Sativa L.) Crops? Eur. J. Agron. 2007, 27, 154–164.
44. Sivesind, E.; Seguin, P. Effects of the Environment, Cultivar, Maturity, and Preservation Method on Red Clover Isoflavone Concentration. J. Agric. Food Chem. 2005, 53, 6397–6402.
45. Booth, N.L.; Overk, C.R.; Yao, P.; Totura, S.; Deng, Y.; Hedayat, A.S.; Bolton, J.L.; Pauli, G.F.; Farnsworth, N.R. Seasonal Variation of Red Clover (Trifolium Pratense L., Fabaceae) Isoflavones and Estrogenic Activity. J. Agric. Food Chem. 2006, 54, 1277–1282.
46. Bhagwat, S.; Haytowitz, D.B.; Holden, J.M. USDA Database for the Isoflavone Content of Selected Foods, Release 2.0. Maryl. US Dep. Agric. 2008, 15, 21.
47. Mazur, W.M.; Duke, J.A.; Wähälä, K.; Rasku, S.; Adlercreutz, H. Isoflavonoids and Lignans in Legumes: Nutritional and Health Aspects in Humans. J. Nutr. Biochem. 1998, 9, 193–200.
48. Caldwell, C.R.; Britz, S.J.; Mirecki, R.M. Effect of Temperature, Elevated Carbon Dioxide, and Drought during Seed Development on the Isoflavone Content of Dwarf Soybean [Glycine Max (L.) Merrill] Grown in Controlled Environments. J. Agric. Food Chem. 2005, 53, 1125–1129.
49. Petrović, M.; Sokolović, D.; Babić, S.; Vymyslický, T.; Marković, J.; Zornić, V.; Dajić-Stevanović, Z. Isoflavones of the Red and Hungarian Clover and Possible Impact on Animal Diet. Czech J. Food Sci. 2021, 39, 169–175.
50. Papadopoulos, Y.A.; Tsao, R.; McRae, K.B.; Mellish, A.E.; Fillmore, S.A.E. Genetic Variability of Principal Isoflavones in Red Clover. Can. J. Plant Sci. 2006, 86, 1345–1347.
51. Chennupati, P.; Seguin, P.; Chamoun, R.; Jabaji, S. Effects of High-Temperature Stress on Soybean Isoflavone Concentration and Expression of Key Genes Involved in Isoflavone Synthesis. J. Agric. Food Chem. 2012, 60, 12421–12427.
52. Kim, S.; Jung, W.; Ahn, J.; Kim, J.; Chung, I. Quantitative Analysis of the Isoflavone Content and Biological Growth of Soybean (Glycine Max L.) at Elevated Temperature, CO2 Level and N Application. J. Sci. Food Agric. 2005, 85, 2557–2566.
53. Reddy, P.S.; Chakradhar, T.; Reddy, R.A.; Nitnavare, R.B.; Mahanty, S.; Reddy, M.K. Role of Heat Shock Proteins in Improving Heat Stress Tolerance in Crop Plants BT - Heat Shock Proteins and Plants. In; Asea, A.A.A., Kaur, P., Calderwood, S.K., Eds.; Springer International Publishing: Cham, 2016; pp. 283–307 ISBN 978-3-319-46340-7.
54. Tsai, C.; Harding, S.A.; Tschaplinski, T.J.; Lindroth, R.L.; Yuan, Y. Genome‐wide Analysis of the Structural Genes Regulating Defense Phenylpropanoid Metabolism in Populus. New Phytol. 2006, 172, 47–62.
55. Weisshaar, B.; Jenkins, G.I. Phenylpropanoid Biosynthesis and Its Regulation. Curr. Opin. Plant Biol. 1998, 1, 251–257.