Effects of High Temperature Stress at Different Development Stages

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Effects of High Temperature Stress at Different Development Stages on Soybean Isoflavone and Tocopherol Concentrations Pratyusha Chennupati, Philippe Seguin,* and Wucheng Liu Department of Plant Science, McGill University, Macdonald Campus, 21111 Lakeshore Road, Sainte-Anne-de-Bellevue, QC H9X 3V9, Canada ABSTRACT: Soybean contains a range of compounds with putative health benefits including isoflavones and tocopherols. A study was conducted to determine the effects on these compounds of high temperature stress imposed at specific development stages [i.e., none, pre-emergence, vegetative, early reproductive (R1 4), late-reproductive (R5 8), or all stages]. Two cultivars (AC Proteina and OAC Champion) were grown in growth chambers set at contrasting temperatures [i.e., stress conditions of 33/25 °C (day/night temperature) and control conditions of 23/15 °C] in order to generate these treatments. Isoflavone and tocopherol concentrations in mature seeds were determined using high-performance liquid chromatography. In both cultivars isoflavone response was greatest when stress occurred during the R5 8 stages and during all development stages, these treatments reducing total isoflavone concentration by an average of 85% compared to the control. Stress imposed at other stages also affected isoflavone concentration although the response was smaller. For example, stress during the vegetative stages reduced total isoflavones by 33% in OAC Champion. Stress imposed pre-emergence had an opposite effect increasing daidzein concentration by 24% in AC Proteina. Tocopherol concentrations were affected the most when stress was imposed during all stages of development, followed by stress restricted to stages R5 8; response to stress during other stages was limited. The specific response of tocopherols differed, α-tocopherol being increased by high temperature by as much as 752%, the reverse being observed for δ-tocopherol and γ-tocopherol. The present study demonstrates that while isoflavone and tocopherol concentrations in soybeans are affected the most by stress occurring during seed formation, concentrations can also be affected by stress occurring at other stages including stages as early as pre-emergence. KEYWORDS: soybean, health-beneficial compounds, isoflavones, tocopherols, stress

’ INTRODUCTION Soybean [Glycine max (L.) Merr] contains several compounds, including isoflavones and tocopherols, that have putative health-beneficial properties .1 Soybean isoflavones are presumed to lessen discomfort caused by menopause and reduce the incidence of certain cancers, cardiovascular diseases, osteoporosis, diabetes, and renal diseases.2 7 Health beneficial properties have also been attributed to tocopherols, α-tocopherol in particular as it is the biologically active form of vitamin E.8 Tocopherols are antioxidants that have the capacity to reduce the incidence of certain diseases including colon cancer.9 Because of these properties, soybean and its extracts have thus become widely used in a range of commercial functional foods and nutraceutical products.10 Several studies have reported that environmental and genotype  environment effects are responsible for most of the variation observed in isoflavone and tocopherol concentrations. Total isoflavone and tocopherol concentrations of a given cultivar may vary by up to 325 and 78% across environments, respectively.11 14 In the case of α-tocopherol, the variation can be as high as 670%.11,14 Such variations are problematic as they hinder functional food development, concentrations being highly inconsistent and unpredictable. Identifying the environmental factors affecting concentrations the most is thus crucial for the industry. Abiotic factors such as air temperature, soil moisture levels, soil fertility (i.e., potassium), CO2 levels, and light quality have been reported to affect soybean isoflavone concentrations; the r 2011 American Chemical Society

relative contribution of specific factors to variations in isoflavone concentrations remains, however, poorly understood.15 Most of the research to date has focused on the effects of periodic stresses, mainly high air temperature and low soil moisture during seed development (R5 7 stages) due to the common assumption that isoflavone accumulation in seeds is only the result of local synthesis.16 19 However, key genes of the phenylpropanoid pathway leading to isoflavone synthesis are expressed in all organs throughout plant development, and translocation of isoflavones into seeds has been reported.20 22 Furthermore, it was recently reported that air temperature prior to emergence is strongly correlated with total isoflavone concentrations in mature seeds, thus underlining the importance to study the impact of abiotic factors at stages other than R5 7.23 In the case of soybean tocopherols, only few studies investigated the impact of abiotic factors on concentrations.24,25 Britz and Kremer26 for example reported that total tocopherol concentration in mature soybean seeds is only slightly affected by elevated temperature and moisture stresses; however, they cause an increase in the proportion of α-tocopherol. These studies focused only on the impact of air temperature and soil moisture on tocopherol concentrations, again during the R5 7 stages. Received: September 16, 2011 Accepted: November 18, 2011 Revised: November 15, 2011 Published: November 18, 2011 13081

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Figure 1. Isoflavone concentrations in mature seeds of two soybean cultivars after exposure to high temperature stress [i.e., 33/25 °C (day/night temperature)] at specific stages of development. Control conditions consisted of day/night temperatures of 23/15 °C. Means for a given cultivar followed by different letters are significantly different (P < 0.05).

The objective of the present study was to determine the impact of elevated temperature stress occurring at various stages of development on soybean isoflavone and tocopherol concentrations, with the goal of identifying the most responsive stages.

’ MATERIALS AND METHODS Experiment and Treatment Description. An experiment was conducted twice in controlled environment growth chambers (model no. E15, Conviron, Winnipeg, MB, Canada). Two soybean cultivars with contrasting isoflavone concentrations, namely, AC Proteina (a highisoflavone cultivar) and OAC Champion (a low-isoflavone cultivar), were used.13 Six seeds of each cultivar were sown in plastic pots with a 3 L capacity filled with a mixture of pasteurized soil, sand, peat, and perlite in a ratio of 2:1:1:1. A commercial (Nitragin, EMD Crop Bioscience, Wisconsin, USA) peat based rhizobial inoculant (Bradyrhizobium japonicum) was added to each pot to ensure nitrogen fixation. Plants were thinned down to two plants per pot two weeks after seeding.

Plants were grown with a 16 h photoperiod which was achieved by using Philips 65 W fluorescence tubes (Philips Electronics, London, ON, Canada) and 60 W incandescent bulbs (Tungsram, Oakville, ON, Canada) with a light intensity of 260 μmol m 2 s 1. Growth chambers were set at contrasting temperatures to provide stress [i.e., 33/25 °C (day/night temperature)] and control conditions (i.e., 23/15 °C). Six treatments were evaluated to expose plants to stress conditions at different stages of development including (i) plants grown during all development stages at 23/15 °C, (ii) plants grown during all development stages at 33/25 °C, (iii) plants grown at 23/15 °C for all stages except pre-emergence during which they were grown at 33/25 °C, (iv) plants grown at 23/15 °C for all stages except for vegetative stages following emergence during which they were grown at 33/25 °C, (v) plants grown at 23/15 °C for all stages except stages R1 4 (flowering to early seed formation)19 during which they were grown at 33/25 °C, and (vi) plants grown at 23/15 °C for all stages except stages R5 8 (seed formation to maturity) during which they were grown at 33/25 °C. The number of days required to reach each specific stage varied depending on the treatment. Emergence was completed 4 to 6 days after seeding, stage 13082

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Journal of Agricultural and Food Chemistry R1 23 to 38 days after seeding, stage R5 38 to 63 days after seeding, and harvest occurred 76 to 90 days after seeding. Plants were watered as needed, and relative humidity was maintained at a similar level in all growth chambers. Seeds were harvested when plants reached full maturity to determine seed yield, the 100-seed weight, and isoflavone and tocopherol concentrations in seeds from the different treatments. Seeds were stored at room temperature for one month and then were finely ground using a coffee grinder (Black & Decker, Towson, MD, USA). Isoflavone Extraction and Quantification. Isoflavone extraction was conducted using a modified version of the protocol of the AOAC official method.27 One hundred milligrams of finely ground soybean seeds were supersonicated in 4.6 mL of 70% aqueous methanol (with 50 μg mL 1 of apigenin as internal standard) for 20 min at room temperature. Further extraction was carried out by shaking for 60 min at 25 °C with orbital shaking at a speed of 200 rpm after the addition of 300 μL of 2 M sodium hydroxide. Neutralization was achieved by the addition of 100 μL of glacial acetic acid. This was followed by centrifugation for 10 min at 10000g; 1.0 mL of the supernatant was then transferred to HPLC vials. Separation of isoflavones was carried out using a Varian system (Walnut Creek, CA, USA) equipped with a Prostar 210 solvent delivery system, a model 410 autosampler and a Prostar 330 photodiode array detector (PDA). Twenty microliter volumes of the extract were used for analyses. Separation was performed on a C18 reversed-phase column (Luna, 5 μm, 4.6  250 mm; Phenomenex, Torrance, CA, USA). The flow rate and the temperature of the column were maintained constant throughout the analyses (i.e., 0.65 mL min 1 and 40 °C). Isoflavones were detected at 260 nm. Mobile phase solvents, 0.05% phosphoric acid (mobile phase A) and HPLC grade acetonitrile (mobile phase B) were used. Isoflavone elution was carried out using a linear gradient system from 10% solvent B, with no hold time after injection, to 30% solvent B over the course of 60 min, followed by 3 min wash with 90% solvent B and 10 min for equilibration with 10% of solvent B. Calibration curves were prepared using purified isoflavones [daidzein, glycitein, genistein, daidzin, glycitin, and genistin (Indofine, Hillsborough, NJ, USA)] as standards. Concentrations of all the isoflavones detected were expressed on a dry matter (DM) basis. Isoflavone concentrations were expressed as aglycon equivalent (i.e., daidzein, glycitein and genistein). Total isoflavone concentration was obtained by summing the concentrations of individual isoflavones. Tocopherol Extraction and Quantification. Tocopherol extraction was performed using a modified version of the protocol of Kitamura et al.28 Supersonication of 50 mg of finely ground soybean seeds was carried out for 15 min at room temperature in 80% aqueous ethanol (with 5 μg mL 1 of tocol as internal standard). The samples were made to stand for 30 min at room temperature after the addition of 1 mL of hexane saturated with pyrogallol. Centrifugation was carried out at 10000g for 10 min. The supernatant (i.e., the hexane layer) was separated and evaporated overnight. The extraction was completed by the addition of 80% aqueous ethanol and high speed vortexing for few seconds. Two hundred microliters of the extract was transferred to HPLC vial. Separation of the tocopherols was done using a Varian system (Walnut Creek, CA, USA) with a Prostar 210 solvent delivery system, a model 410 autosampler, and a Prostar 325 UV detector. Twenty microliters of the extract was used to carry out the separation on an Inertsil ODS-3 reverse phase column (5 μm, 3.0  250 mm; GL Science, Japan). A flow rate of 0.5 mL min 1 was used. Two mobile phases CH3CN/CH3OH in the ratio of 75:25 (v/v) were used. The run time of each sample was 25 min. Tocopherols were detected at 295 nm. Calibration curves were plotted using γ-, δ-, and α-tocopherol standards (Sigma Aldrich, St. Louis, MO, USA). Quantification of individual tocopherols was done based on the resulting curves. Individual tocopherol concentration was summed up to obtain the total tocopherol

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concentration. β-Tocopherol, which is found in very small concentrations in soybean seeds [i.e., 90%) when stress was imposed during all development stages, followed by stress restricted to the R5 8 stages. Stress imposed during the vegetative stages or during the R1 4 stages affected isoflavone concentrations the least, resulting in average reductions of 25%. As for AC Proteina, 13083

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Figure 2. Contribution of each isoflavone to total isoflavone concentration in mature seeds of two soybean cultivars after exposure to high temperature stress [i.e., 33/25 °C (day/night temperature)] at specific stages of development. Control conditions consisted of day/night temperatures of 23/15 °C. Means for a given cultivar followed by different letters are significantly different (P < 0.05).

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the contribution of each individual isoflavone to total isoflavones was changed by specific treatments when compared to the control; however, the response was different. Stress imposed during the vegetative stage or during the R5 8 stages increased the percentage of daidzein and glycitein, but reduced that of genistein. Stress imposed during all development stages only reduced the percentage of daidzein. Tocopherols. Both cultivars responded differently to stress treatments, as indicated by significant cultivar  stress treatment interactions for α- and δ-tocopherol (P < 0.05). However, in the case of α-tocopherol, the interaction reflected differences in the magnitude of the response of both cultivars. Independent of this fact, the greatest response to stress treatments was observed for α-tocopherol. For AC Proteina, when compared to the control, α-tocopherol concentration was increased by 752 and 675% when stress was imposed during all development stages or restricted to the R5 8 stages, respectively (Figure 3). For OAC Champion, the response to these two treatments was smaller, concentrations being increased by 341 and 441% when stress was imposed during all development stages or restricted to the R5 8 stages, respectively. Stress failed to impact α-tocopherol concentration in both cultivars when it was imposed during other development stages. In contrast, γ-tocopherol concentration was reduced by an average of 34% in both cultivars compared to the control, only when stress occurred during all development stages. For AC Proteina, the response of δ-tocopherol was also the opposite of that of α-tocopherol. Stress imposed during the R5 8 stages and during all development stages reduced concentration by 66 and 74% respectively. Stress restricted to vegetative stages also decreased δ-tocopherol concentration although the decrease was only 7%. For OAC Champion, stress imposed during the R5 8 stages and during all development stages reduced concentration by 61 and 73%, respectively. Stress restricted to the R1 4 stages also decreased δ-tocopherol concentration in that cultivar but by only 9%. Finally, total tocopherol concentration was reduced in both cultivars by an average of 22% by stress occurring during all development stages. Overall, across stress treatments, total and α-tocopherol concentrations were greater for AC Proteina than OAC Champion; both cultivars had however comparable concentrations of γ- and δ-tocopherol. The contribution of each individual tocopherol to the total tocopherols concentration was changed by specific treatments when compared to the control (Figure 4). Stress imposed during all development stages increased the proportion of α-tocopherol (i.e., to 43 and 28%, in AC Proteina and OAC Champion, respectively); stress restricted to the R5 8 stages also increased this contribution but to a lower extent (i.e., to 33 and 24% in AC Proteina and OAC Champion). This difference is attributable to the fact that while both treatments similarly increased α-tocopherol concentration, only stress imposed during all development stages also decreased total tocopherol concentration. The proportions of both γ- and δ-tocopherol were also decreased in both cultivars by stress imposed during all development stages. A similar response to stress restricted to the R5 8 stages was also observed for δ- and γ-tocopherol, although the response was more moderate for γ-tocopherol. Finally, stress restricted to the vegetative or the R1 4 stages slightly decreased the proportion of δ-tocopherol and increased that of γ-tocopherol, when compared to the control. Seed Yield and 100-Seed Weight. The yield response of both cultivars to temperature treatments differed as indicated by significant cultivar  stress treatment interactions (P < 0.05). 13084

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Figure 3. Tocopherol concentrations in mature seeds of two soybean cultivars after exposure to high temperature stress [i.e., 33/25 °C (day/night temperature)] at specific stages of development. Control conditions consisted of day/night temperatures of 23/15 °C. Means for a given cultivar followed by different letters are significantly different (P < 0.05).

For AC Proteina, seed yield per plant was reduced by 21% compared to the control when the stress treatment was imposed during all development stages, transient stress failing to impact seed yield (Figure 5). The 100-seed weight was reduced by all stress treatments except for those imposed prior reproductive stages. The reduction ranged between 12 and 32% and was greatest when stress was restricted to the R5 8 stages. For OAC Champion, compared to the control, seed yield per plant was reduced by stress treatments imposed during all development stages (i.e., by 33%) and during the R1 4 stages (i.e., by 23%). Stress treatments imposed during all development stages and during the R5 8 stages reduced the 100-seed weight by 16 and 18% respectively, when compared to the control. It was, however, increased by 20% when stress was restricted to the vegetative stages. Finally, seed yield per plant across treatments was greater for OAC Champion than AC Proteina. This reflected the overall greater 100-seed weight of OAC Champion compared to AC Proteina, as both cultivars had comparable number of pods per plant and number of seeds per pod (data not presented).

’ DISCUSSION The isoflavone response to a high temperature stress imposed during the R5 8 stages observed herein confirms results from

previous studies. The response of soybean to a high temperature stress at this particular stage of development is indeed well documented, most studies reporting that a 10 to 15 °C increase in air temperature during seed development results in a 3- to 6-fold reduction in total isoflavone concentration of mature seeds.16 18,31 The present study is, however, the first to directly report on the impact of a high temperature stress at other stages of development. While the most responsive stages remain the R5 8 stages, stress imposed at other stages will also affect isoflavone concentrations in mature seeds. Stress imposed from stage R1 to 4 consistently reduced concentrations of individual and total isoflavones, although the magnitude of the response depended on the cultivar (Figure 1). Stress imposed during vegetative stages also consistently reduced concentrations of all isoflavones in OAC Champion but only of genistein in AC Proteina. The present study is also the first to report directly that high temperature stress occurring pre-emergence may increase isoflavone concentration in soybean, a response observed for daidzein in AC Proteina. In accordance with these results, a correlation between air temperature pre-emergence and isoflavone concentrations in mature seeds was previously reported from field studies by Morrison et al.23 The present results confirm that the response of soybean seed isoflavones to temperature 13085

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Figure 5. Seed weight per plant and 100-seed weight of two soybean cultivars after exposure to high temperature stress [i.e., 33/25 °C (day/night temperature)] at specific stages of development. Control conditions consisted in day/night temperatures of 23/15 °C. Means for a given cultivar followed by different letters are significantly different (P < 0.05).

Figure 4. Contribution of each tocopherol to total tocopherol concentration in mature seeds of two soybean cultivars after exposure to high temperature stress [i.e., 33/25 °C (day/night temperature)] at specific stages of development. Control conditions consisted of day/night temperatures of 23/15 °C. Means for a given cultivar followed by different letters are significantly different (P < 0.05).

is complex not being restricted to events occurring during seed formation. It is hypothesized that the response of soybean

isoflavones to high temperature reflects an impact of temperature on the expression of key genes involved in isoflavone synthesis. Other stresses including soil moisture deficits have indeed been reported to downregulate the expression of key genes involved in isoflavone synthesis.32 Results from the present study for tocopherols confirm previous ones which reported that increases in temperature during the R5 7 stages increase α-tocopherol concentration.29 As for isoflavones, the present study is, however, the first one to report that high temperature stress at other stages can also affect soybean tocopherol concentrations. The degree of response depended on the cultivar, the development stage at which the stress was imposed, and the tocopherol form. Overall, tocopherol concentrations were affected the most when stress was imposed during all stages of development, followed by stress restricted to stages R5 8. The response of specific tocopherols although differed, with α-tocopherol being increased by high temperature, 13086

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Journal of Agricultural and Food Chemistry the reverse being observed for δ-tocopherol and γ-tocopherol. This opposite response of δ-tocopherol and γ-tocopherol suggests an effect of temperature on the enzyme γ-tocopherol methyl transferase or the gene responsible for its synthesis. Indeed, α-tocopherol is synthesized from γ-tocopherol through its methylation by γ-tocopherol methyl transferase; this same enzyme also mediates the synthesis of β-tocopherol from δ-tocopherol.33 While monitoring seed yield response to high temperature was not a key objective of the current project, studies looking at the impact of abiotic and biotic factors on crop composition should also determine the impact of treatments on seed yield and size, as these are two important factors in determining the profitability of any production. In the present study it appeared that treatments that increased α-tocopherol the most also negatively affected seed yield and/or the 100-seed weight. In conclusion, the present study is the first one to directly report on the effects of high temperature stress at specific stages of development on soybean isoflavone and tocopherol concentrations. It demonstrated clearly that isoflavone and tocopherol concentrations in mature seeds can be affected by stress occurring at stages other than seed formation (i.e., R5 7), including stages as early as pre-emergence. However, given the differential response of cultivars observed in the present study, it will be essential to evaluate the response of a larger number of cultivars before major conclusions can be formulated. Results observed suggest that temperature could affect isoflavone and tocopherol concentrations in one or several of 3 ways: (i) by degrading or transforming these compounds or some precursors, (ii) by changing their translocation rate toward seeds and/or toward other organs, or (iii) by altering expression of key genes involved in their synthesis. More controlled experimentation including gene expression studies will be required to test these hypotheses.

’ AUTHOR INFORMATION Corresponding Author

*Tel: 1-514-398-7855. Fax: 1-514-398-7897. E-mail: philippe. [email protected]. Funding Sources

This research was supported in part by a Discovery Grant from the Natural Sciences and Engineering Research Council of Canada (NSERC).

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