Published online 26 August 2005
Published in Crop Sci 45:1934-1940 (2005)
© 2005 Crop Science Society of America
677 S. Segoe Rd., Madison, WI 53711 USA
CROP PHYSIOLOGY & METABOLISM
Effect of Temperature and Soil Moisture Status during Seed Development on Soybean Seed Isoflavone Concentration and Composition
Vera V. Lozovayaa,*,
Anatoliy V. Lygina,
Alexander V. Ulanova,
Randall L. Nelsonc,
Jean Daydéb and
Jack M. Widholma
a Dep. of Crop Sciences, 1201 W. Gregory Dr., Univ. of Illinois, Urbana, IL 61801, USA
b Ecole Supérieure d'Agriculture de Purpan, 75 voie du TOEC, 31076 Toulouse Cédex 03, France
c USDA-ARS, Soybean/Maize Germplasm, Pathology, and Genetics Research Unit, Dep. of Crop Sciences, 1101 W. Peabody Dr., Univ. of Illinois, Urbana, IL 61801, USA
* Corresponding author (lozovaya{at}uiuc.edu)
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ABSTRACT
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Soybean [Glycine max (L.) Merr.] seed isoflavone concentration has been shown to be highly dependent on environmental conditions, but isoflavone concentrations have not been studied under controlled conditions to quantify the effects of specific factors. To determine the effect of air temperature and soil moisture status during soybean seed development on seed isoflavone concentration and composition, soybean plants were grown in the greenhouse under intermediate (18/28°C), 9.5 h night/14.5 h daytime temperatures with high soil moisture conditions. Beginning at the R6 growth stage plants were subjected to either intermediate (18/28°C), low (13/23°C), or high (23/33°C) 9.5 h night/14.5 h daytime temperatures with either low or high soil moisture conditions. Two French cultivars, Imari and Queen, and three U.S. cultivars, Dwight, Jack and Loda, all in maturity group II were studied. The overall results show that low temperatures and high soil moisture conditions produced the highest seed isoflavone concentrations with changes in temperature having the larger effect. The changes in daidzein and genistein concentrations were similar to changes in total isoflavones but the glycitein concentration was much less affected. The three U.S. cultivars were much less responsive to soil moisture than the two French cultivars. All five cultivars showed a two- to threefold increase in total isoflavone concentrations at the low temperature regime compared to the high temperature regime. Plant height was greatest under the intermediate temperatures; whereas, low temperatures and low soil moisture hastened maturity. Seed size was not significantly affected by any treatment. Soil moisture and air temperature have clear effects on the isoflavone concentrations in mature soybean seeds, but the ranking of all the cultivars based on average isoflavone concentration remained the same with all treatments. Environmental factors can have a large effect on isoflavone concentration, but the potential for isoflavone production is largely under genetic control.
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INTRODUCTION
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SOYBEAN SEED ISOFLAVONES have been shown to have beneficial effects on human health. Studies comparing populations in southeast Asia, where soy consumption is high, and the rest of the world, as well as other scientific research, have indicated that soy isoflavones are beneficial for decreasing certain cancers, osteoporosis, cardiovascular disease, and menopausal symptoms (Anderson and Garner, 1997; Murkies et al., 1998; Setchell, 1998; Anderson et al., 1999; Setchell and Cassidy, 1999). Studies with humans (Crouse et al., 1999) and monkeys (Anthony et al., 1997) show that isoflavone-rich soy protein isolates are more effective than purified isoflavones for lowering blood cholesterol. The soybean isoflavones daidzein, genistein, and glycitein occur predominantly as glucosides or malonylglucosides (Ohta et al., 1979) and are associated with the protein fraction during processing. Due to the health benefits there is an increased interest in soy foods in the USA and other parts of the world.
Several studies have shown that there is a large variation in isoflavone concentration and composition among soybean genotypes (Eldridge and Kwolek, 1983; Kitamura et al., 1991; Wang and Murphy, 1994; Choi et al., 1996; Hoeck et al., 2000; Nelson et al., 2001). The seed isoflavone concentrations are also affected by year and location indicating a large environmental effect (Eldridge and Kwolek, 1983; Wang and Murphy, 1994; Lacombe et al., 1999; Hoeck et al., 2000). Kitamura et al. (1991) and Tsukamoto et al. (1995) reported a negative correlation between isoflavone concentrations and temperature during seed development. These results are consistent with the findings that soybeans planted later in the growing season (Aussenac et al., 1998) and later maturing lines (Nelson et al., 2001) usually have higher seed isoflavone concentrations. Previous research has not attempted to quantify the effects of temperature by growing plants under controlled environmental conditions nor has any previous published research investigated the role of the soil moisture or the interaction of air temperature and soil moisture on isoflavone concentration. The objective of our research was to evaluate the effects of air temperature and soil moisture conditions during soybean seed fill on seed isoflavone concentrations of soybean cultivars with known differences in isoflavone concentration.
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MATERIALS AND METHODS
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Two French soybean cultivars, Imari and Queen, and three U.S. cultivars, Dwight, Jack, and Loda, were selected based on similar times of maturity, large differences in isoflavone concentrations, and preliminary data that indicated differential responses to environmental changes. All entries were grown one plant per 30-cm diameter plastic pot under intermediate night/daytime temperatures of 18/28°C (23°C mean) with high soil moisture in the Plant Sciences Laboratory greenhouses on the campus of the University of Illinois (Urbana, IL). A sand/soil/perlite (1:1:1) mixture was used and plants were fertilized by watering with 20–20–20 (N–P–K; 250 mg kg–1 each) with micronutrients (Plant Marvel Laboratories Inc., Chicago Heights, IL). Plants were grown under a 14.5-h photoperiod that was maintained with the use of 1000-W high pressure sodium lamps for lighting (600 W m–2). When the plants reached the R6 stage, they were moved into one of three different temperature regimes until maturity: (i) intermediate (as above), (ii) low 13/23°C (18°C mean), and (iii) high 23/33°C (28°C mean). In each temperature treatment, one-half of the plants were grown in high soil moisture (approximately 70% of soil holding capacity) and one-half in low soil moisture (approximately 30% of the high treatment as measured with a HydroSense Soil Water Measurement System device, SC 620 [Spectrum Technologies, Inc., Plainfield, IL]). Soil moisture was measured and adjusted every morning. Maturity date, plant height, seed yield per plant, and 100 seed weight were measured. Isoflavone concentrations were measured in mature seed samples from each plant. The experiment was repeated twice.
Isoflavone Extraction and Analysis
Twenty-five soybean seeds of each sample were ground for 5 min with a mechanical mill (Spex Industries, Inc., Metuchen, NJ), and 200 mg of the powder was extracted with 1 mL of 80% (v/v) methanol at 20°C with vigorous shaking (80 rpm) in a G10 gyrotary shaker (New Brunswick Sci., Edison, NJ) for 12 h. The pellet obtained after centrifugation at 5000 g for 10 min was extracted again with 1 mL 80% methanol by shaking for 2 h and samples were centrifuged at 5000 g for 10 min. The combined supernatants (2 mL) were centrifuged at 12000 g for 10 min and then used for HPLC analysis.
Isoflavones were separated with a Waters HPLC system (Waters Corp., Milford, MA) using a 53 by 7 mm, 3 µm EPS C18 Alltech Rocket Column (Alltech Assoc., Deerfield, IL) following a previously reported method (Graham, 1991). A linear gradient composed of water (solvent A) (pH 2.8, adjusted with acetic acid) and acetonitrile (solvent B) was used. Following injection of 20 µL of sample, solvent B was increased from 5 to 12% over 7 min and then isocratic elution for 3 min occurred followed by an increase in solvent B to 35% over 15 min. The column was then washed with 95% acetonitrile for 3 min and equilibrated for 3 min at 5% B between runs. Total sample to sample time was 30 min. The solvent flow was 2.5 mL min–1. A Waters 996 photodiode array detector was used to measure UV absorbance at 253 nm. Isoflavone standards (daidzein, genistein, glycitein, and their glucosides) were purchased from LC Laboratories (Woburn, MA). The concentrations of malonyl forms were determined based on the curves for the corresponding ß-glucosides and appropriate corrections for the molecular mass differences since the molar extinction coefficient of the esterified isoflavones are similar to that of the ß-glucosides (Barnes et al., 1994). Each sample was analyzed in two replicates. Standard errors between the same samples did not exceed 2%. Isoflavone concentrations were reported as microgram of the aglycone form per gram of seed dry weight.
Statistical Analysis
The experiment was planted in a completely randomized design with five replications. The entire experiment was repeated twice. Analysis of variance was used to partition the total variance for all variables measured using PC SAS (SAS Institute, 1999). Cultivars and treatments were considered fixed in the model. Significant differences were declared using protected least significant differences calculated with PROC ANOVA (SAS Institute, 1999).
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RESULTS AND DISCUSSION
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Cultivars
Significant differences were found among the cultivars for all traits measured (Table 1), but none of the agronomic data was predictive of isoflavone concentrations (Table 2). Dwight and Queen, among the latest and earliest maturing entries, respectively, had the highest concentrations of daidzein, genistein, and total isoflavones. Imari had the lowest genistein, glycitein, and total isoflavone concentrations but was not significantly lower than Jack for daidzein concentration. Queen had the highest concentration of glycitein. Averaged over 60 observations for each cultivar, these highly significant differences confirm that large genetic differences do exist for isoflavone concentrations and these differences are maintained over very diverse environmental conditions.
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Table 1. Analysis of variance of isoflavone concentration and agronomic characters measured for five soybean cultivars grown under differing temperature and soil moisture conditions in a greenhouse experiment.
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Table 2. Entry means for isoflavone concentrations and other plant data averaged over five replications per experiment, two experiments, three air temperature regimes, and two soil moisture treatments.
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Temperature
The treatments were imposed at the R6 growth stage to reduce the effects on overall plant development; however, there were significant temperature effects for maturity, height, and yield per plant but perhaps most importantly for this research not for 100 seed weight (Table 1). Maturity was delayed with increased temperature (Table 3). The plants grown in the intermediate temperatures regardless of the soil moisture treatment conditions were significantly taller than those grown in either of the other temperature regimes. Each incremental decrease in temperature significantly increased daidzein, genistein, and total isoflavone concentrations. Glycitein was less strongly affected and the only significant change occurred between the high and low temperature treatment (Table 3). Lowering from high temperatures to intermediate temperatures increased total isoflavone concentration by a highly significantly 48%, but lowering from intermediate to low temperatures changed total isoflavone concentration by 69% (Table 3). Even larger increases were observed with daidzein with changes of 65 and 82% when making the same comparisons (Table 3). The response for genistein was similar to that of total isoflavones with changes of 48 and 74%. Temperature not only changed the amount but can also change the composition of seed isoflavones. For these cultivars at high temperatures, daidzein was 35% of the total; genistein was 48%; and glycitein was 16%. At low temperatures, daidzein increased to 42% and genistein to 50%, but glycitein was only 8% of the total.
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Table 3. Effects of temperature and soil moisture on isoflavone concentration, plant maturity and height, and seed yield and weight averaged over five cultivars, five replications per experiment, and two experiments.
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Soil Moisture
Averaging across all temperatures demonstrates that imposing limited soil moisture at R6 growth stage significantly hastened maturity and reduced yield per plant but did not change weight per seed nor plant height (Table 1). These data indicate that the treatments had very little impact on seed dry matter accumulation and any changes noted in isoflavone concentration are very likely to be the direct result of changes in soil moisture or air temperature and not artifacts of changes in seed dry matter. Soil moisture effects on seed isoflavones were significant but less pronounced than those of temperature (Table 3). High soil moisture increased the concentrations of daidzein, genistein, and total isoflavones but did not significantly change the glycitein concentrations (Table 3).
Treatment Combinations
There were highly significant differences for total isoflavone concentration among five of the six treatment combinations (Table 4). As would be expected from the treatment main effects, the highest concentrations were produced under the lowest temperatures and the lowest concentrations were produced under the highest temperatures. Within these two temperature regimes, lowering soil moisture also significantly reduced total isoflavone concentration (Table 4). Intermediate temperatures produced intermediate levels of total isoflavones but lowering soil moisture at intermediate temperatures did not significantly reduce total isoflavone concentrations (Table 4). The response patterns observed for total isoflavones were identical for both daidzein and genistein concentrations. The environmental effects were not significant for glycitein concentration.
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Table 4. Effects of temperature and soil moisture combinations on isoflavone concentration, plant maturity and height, and seed yield and weight averaged over five cultivars, five replications per experiment, and two experiments.
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Cultivar x Soil Moisture Interactions
Examining the effects of soil moisture on isoflavone concentrations in individual cultivars reveals a significant cultivar x soil moisture interaction for all isoflavone values except for glycitein concentration (Table 1). None of the U.S. cultivars had a significant change in total isoflavone concentration due to changes in soil moisture and most of the values were nearly identical across treatments (Table 5). The two French cultivars had very large and highly significant increases in total isoflavones as well as in daidzein and genistein concentrations with increases in soil moisture (Table 5). The changes in the U.S. cultivars for total isoflavones between the high and low soil moisture were between 3 and 4% with some responses positive and some negative. Imari increased 55% and Queen increased 42%. Although the total isoflavone concentration of Loda did not change between soil treatments, genistein was significantly reduced with lower soil moisture (Table 5).
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Table 5. Entry by soil moisture interactions for isoflavone concentration, plant maturity and height, and seed yield and weight averaged over five cultivars, five replications per experiment, and two experiments.
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Cultivar x Temperature Interactions
The effects of temperature were more uniformly expressed across all cultivars but a significant cultivar x temperature interaction was detected for all isoflavone concentrations except for glycitein. (Table 1). Differences among all temperature treatments for total isoflavones were significant except for the differences between the high and intermediate temperatures for Imari and Loda (Table 6). Queen increased 75% between the high and intermediate temperatures. Total isoflavones for Imari increased 96% between the intermediate and low temperatures but only 51% for Queen; however, the changes observed between these two temperature regimes were highly significant for all cultivars (Table 6). Both Jack and Imari had total increases of more than 200% when comparing the differences between high and low temperatures. Dwight had the lowest total response of only 110%. Since Dwight had a significantly higher concentration of total isoflavones than all entries except for Queen, this lack of response could indicate a greater tolerance for high temperatures. Dwight has numerically higher values than Queen at high temperatures for daidzein, genistein, and total isoflavones although only the genistein difference is statistically significant (Table 6). Queen had consistently larger percentage increases between high and intermediate temperatures than between intermediate and low temperatures and was the only cultivar with this response. This may indicate Queen is more sensitive to high temperatures. The difference between high and intermediate temperatures for daidzein concentration was not significant for Imari, Jack, or Loda even though Jack increased by 84% (Table 6). For genistein, the differences between high and intermediate temperatures were not significant for Loda and Imari (Table 6). This lack of significance reflects the low levels of isoflavones in these entries and not the relative magnitude of the change occurring. All of the differences between the intermediate and low temperatures were significant for daidzein, genistein and total isoflavones (Table 6). As was noted earlier, the composition of isoflavones changed when the temperature was altered but the results were not consistent among cultivars. At the highest temperatures, daidzein was 42% of the total isoflavones in Imari. At the lowest temperatures that percentage was reduced to 40%. In contrast, at the highest temperatures, daidzein was 36% of the total isoflavones in Queen, but at the lowest temperatures that percentage is increased to 50. All but Imari had a reduction in the percentage of daidzein as temperature was increased. The response of genistein was even more variable. Between the highest and lowest temperatures produced, there was little or no change in Dwight and Loda; the percentage of genistein increased in Imari and Jack, and decreased in Queen. The absolute amount of glycitein was not significantly affected by changes in temperature so as the total isoflavones decreased with higher temperatures the percent of glycitein increased. The greatest percentage change was observed in Jack.
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Table 6. Entry by temperature interactions for isoflavone concentration, plant maturity and height, and seed yield and weight averaged over five cultivars, five replications per experiment, and two experiments.
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Cultivar x Temperature x Soil Moisture Interactions
A significant cultivar x temperature x soil moisture interaction was found for daidzein, genistein, and total isoflavone concentration but not for glycitein concentration (Table 1). Based on mean responses, Queen produced the most consistent changes to temperature and soil moisture with an increase with each incremental change to lower temperatures or increased soil moisture. Queen showed changes from 1418 µg g–1 of total isoflavones under hot and dry conditions to 5555 µg g–1 under cool, moist conditions (Table 7). Under optimal conditions, Queen produced the highest level of isoflavones of any entry. Imari also produced the highest isoflavone concentration with the lowest temperatures and highest soil moisture. Total isoflavones in both Imari and Queen increased significantly with increasing soil moisture under the lowest temperature regime. The remaining three cultivars had small nonsignificant decreases in total isoflavone concentrations with this treatment change (Table 7). All cultivars had the lowest isoflavone concentrations with high temperatures and low soil moisture. Based on percentage change, Imari was the most sensitive to environmental changes with nearly a 470% change from the lowest to the highest values. Imari was also significantly lower in total isoflavone concentration than the other entries in most treatments. The greatest absolute change occurred in Queen (4137 µg g–1). The smallest percentage change (134%) and smallest absolute change (1742 µg g–1) induced by environmental conditions was measured in Loda (Table 7).
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Table 7. Entry by temperature by soil moisture interactions for isoflavone concentration, plant maturity and height, and seed yield and weight averaged over five cultivars, five replications per experiment, and two experiments.
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Imari and Queen produced the highest levels of daidzein and genistein at the low temperatures, high soil moisture treatment. Loda, Jack, and Dwight all had reductions in daidzein concentration when the soil moisture was increased at the lowest temperature regime (Table 7). This decrease was significant for Loda. All entries except Jack produced the most genistein at the low temperatures, high soil moisture treatment, and the increases between the low and high soil moisture were statistically significant for Imari and Queen. Percentage and absolute value changes were smallest with glycitein and none of the changes measured were statistically significant (Table 7).
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CONCLUSIONS
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These results are consistent with those of Kitamura et al. (1991) and Tsukamoto et al. (1995) who reported that low temperatures during seed development led to higher soybean seed isoflavone concentrations. This is also consistent with the results of Aussenac et al. (1998) where later planting, which result in lower temperatures during seed fill, also resulted in higher isoflavone concentrations. The higher isoflavone concentrations often found in later maturing genotypes (Nelson et al., 2001) is also consistent with our results obtained under controlled greenhouse conditions. Brevedan and Egli (2003) also showed that water stress during soybean seed fill in greenhouse experiments results in earlier maturity and lower seed yield, but they also found that the seeds were 25 to 33% smaller. The differences in our results where seed size was not changed by the treatments could be due to the differences in genotypes, the water stress conditions used, and the timing of when the stress was applied.
Temperature during seed filling is a major environmental factor influencing isoflavone concentration. In general, lower temperatures increase isoflavone levels but the magnitude of response is cultivar dependent. Soil moisture can also change isoflavone levels with moisture stress reducing isoflavone concentrations, but most of the cultivars that we tested did not show a significant response to reduced soil moisture. Our research clearly demonstrated that the magnitude of changes in seed isoflavone concentrations in response to changes in temperature and soil moisture is highly cultivar dependent. Temperature and soil moisture can differentially affect daidzein, genistein, and glycitein so changes in environmental conditions can change both the amount and composition of total isoflavones.
Even though changes in temperature and soil moisture that we produced in the greenhouse could change the total isoflavone concentration by over 400%, the ranking of cultivars by average isoflavone concentration remained consistent. This shows that isoflavone potential is under strong genetic control. These cultivars and environmental conditions could provide ideal tissues for molecular and biochemical studies to better understand the control of isoflavone biosynthesis in soybean. From a practical application standpoint, to have the highest soybean seed isoflavone concentrations one needs to select the correct cultivar, plant so that seed fill occurs during cool conditions and maintain the soil moisture at adequate levels.
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ACKNOWLEDGMENTS
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This project was carried out with funds from the Illinois-Missouri Biotechnology Alliance, the United Soybean Board, The College of ACES Value-Added Program, the Illinois Soybean Program Operating Board, the Illinois Agricultural Experiment Station and the USDA, Agricultural Research Service.
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NOTES
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Mention of a trademark, proprietary product, or vendor does not constitute a guarantee or warranty of the product by the USDA or the University of Illinois and does not imply its approval to the exclusion of other products or vendors that may also be suitable.
Received for publication September 23, 2004.
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ABSTRACT
INTRODUCTION
