Selenium speciation profiles in selenite-enriched soybean (Glycine Max) by HPLC-ICPMS and ESI-ITMS

Abstract

Soybean (Glycine Max) plants were grown in soil supplemented with sodium selenite. A comprehensive selenium profile, including total selenium concentration, distribution of high molecular weight selenium and characterization of low molecular weight selenium compounds, is reported for each plant compartment: bean, pod, leaf and root of the Se-enriched soybean plants. Two chromatographic techniques, coupled with inductively coupled plasma mass spectrometry (ICPMS) for specific selenium detection, were employed in this work to analyze extract solutions from the plant compartments. Size-exclusion chromatography revealed that the bean compartment, well-known for its strong ability to make proteins, produced high amounts (82% of total Se) of high molecular weight selenospecies, which may offer additional nutritional value and suggest high potential for studying proteins containing selenium in plants. The pod, leaf and root compartments primarily accumulate low molecular weight selenium species. For each compartment, low molecular weight selenium species (lower than 5 kDa) were characterized by ion-pairing reversed phase HPLC-ICPMS and confirmed by electrospray ionizationion trap mass spectrometry (ESI-ITMS). Selenomethionine and selenocystine are the predominant low molecular weight selenium compounds found in the bean, while inorganic selenium was the major species detected in other plant compartments.

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Introduction

Selenium has been increasingly studied in the last two decades. In trace amounts, selenium is an essential micronutrient and has important nutritional benefits for animals and humans;¹ however it can be toxic at high dosages.² The narrow range of beneficial selenium levels, 0.05 to 2 mg kg⁻¹ dry forage feed,³ has important implications for human health. In this respect, plants can play a significant role, for instance, plants that accumulate selenium may be useful to supplement the mammalian diet in geographical areas that are selenium deficient.⁴ Furthermore, some selenium metabolites produced by plants have high nutritional value,⁵ for example, the major selenium metabolite in Se-enriched plants, such as garlic, onions, broccoli florets and sprouts, is Se-methylselenocysteine,⁶ which as reported can be more effective as a cancer chemopreventative than other selenium compounds.⁷ Additionally areas with selenium contamination can be detoxified by growing hyperaccumulating plants in soil or hydroponically to sequester selenium.⁸

Extensive efforts have been devoted to understanding selenium in plants^9,10 by studying the effect of selenium supplementation^11,12 and utilizing Se-enriched plants for health benefits.^13–15 Selenium speciation has been accomplished in a number of plants, from Se-accumulators to cereal grains or vegetables,⁶ but selenium speciation in soybean, one of the most important and widely used commodities, has not been fully explored or understood. Soybean (Glycine Max) is one of the most important food sources worldwide, being consumed directly or made into vegetable oil, soy milk, infant formula, tofu, soy flour, etc. About 40% of its dry weight is composed of proteins, offering all the amino acids essential to human nutrition, and making it a good substitute for animal products.¹⁶

It is well-known that in plants, selenocysteine (SeCys) and selenomethionine (SeMet) can compete and replace cysteine and methionine, respectively, by following sulfur metabolic pathways.^9,17 This non-specific incorporation process produces selenium-containing proteins, in which some sulfur atoms are replaced by selenium. It has been suggested that plants can utilize specific selenium incorporation to make selenoproteins, which are essential to plant’s growth or survival.^18–22 The first direct proof that a UGA opal codon¹ is decoded in the plant kingdom to incorporate selenocysteine was found in Chlamydomonas reinhardtii,¹⁸ indicating the presence of an essential selenoprotein. However, the question of the essentiality of selenium as a micronutrient in plants is not understood, because no essential selenoprotein in higher plants has been clearly identified by protein or DNA sequence analysis. The ability of soybean plant to produce high protein levels, and to accumulate reasonable amounts of selenium, suggest that this may be a model plant to look for selenoproteins.

Since it is widely used, the soybean may also be a good sample to be studied as a dietary selenium source. In addition to providing high quality protein with low saturated fat and high dietary fiber, soybean is a possible source of prevention against cardiovascular diseases and different chronic diseases.¹⁶ A few studies indicate that the low incidence of prostate cancer in Asia may be ascribed to the high consumption of soybeans.²³ As such an important agricultural product, soybean has been intensively studied but few publications have discussed selenium species in soybean, with regard to any increased nutritional value.

SeMet was first identified in soybean about two decades ago, using thin-layer chromatography and gas chromatography-mass spectrometry.^24,25 Hydride-generation atomic fluorescence spectrometry was then used to measure total selenium concentration in Se-enriched soybean.²⁶ The recent study by Yathavakilla and Caruso²⁷ identified a few selenium compounds in the soybean root supplemented with both selenium and mercury using HPLC-ICPMS. Plant roots are important for studying Se–Hg interaction (antagonism) due to the high level of Hg accumulation in the root, but other compartments are of more interest with respect to dietary enhancements, the bean in particular. For example, Se-enriched soybean bean offers the possibility to enhance the concentration of food selenium, making it available to the 0.5 to 1 billion people who suffer from inadequate amounts of selenium in the diet.²⁸

ICPMS, the most readily available and sensitive method of detection for selenium, is an excellent tool for elemental speciation.²⁹ Coupled with HPLC, ICPMS can detect trace amounts of selenium species, even in complex matrices such as blood.³⁰ However, ICPMS cannot lend itself to structural characterization because the highly energetic plasma generates elemental ions, therefore, molecular mass spectrometry, ESI-ITMS, was utilized to compliment ICPMS elemental detection. This study presents a selenium profile for each compartment: bean, pod, leaf and root of the Se-enriched soybean plant. Size exclusion chromatography and ion-pairing reversed phase HPLC, two complementary separation methods, were used to investigate high and low molecular weight selenium species, respectively.

Experimental

Instrumentation

Inductively coupled plasma mass spectrometry . An Agilent 7500ce ICPMS (Agilent Technologies, Santa Clara, CA, USA) was used for selenium detection. It is equipped with an octopole collision/reaction cell and can be operated with or without the collision/reaction gas. A conventional Meinhard nebulizer, a Peltier-cooled spray chamber (2 °C) and a shield torch constitute the sample introduction system under standard plasma conditions. Daily optimization was performed and standard instrument conditions can be found in Table 1.

Table 1 Operating conditions for ICPMS, HPLC and nanoESI-ITMS

ICPMS parameters
Forward power (W)	1500
Plasma gas flow rate (L min^−1)	15.0
Carrier gas flow rate (L min^−1)	0.97
Isotopes monitored	^77Se, ^78Se, ^80Se, ^82Se
Collision gas (mL H2 min^−1)	3.5
Quadrupole bias (V)	−16
Octopole bias (V)	−18
Net energy barrier (V)	+2
SEC chromatographic parameters
Columns	Superdex Peptide 10/300 GL
	Superdex 200 10/300 GL
Mobile phase	30 mM Tris-HCl buffer, pH 7.5
Flow rate (mL min^−1)	0.6
Injection volume (μL)	100
IPRP-HPLC chromatographic parameters
Column	Agilent Zorbax SB-C18
Mobile phase	0.02% HFBA 2% MeOH
Gradient	Isocratic
Flow rate (mL min^−1)	1.0
Injection volume (μL)	50
nanoESI-ITMS parameters
Nanospray needle voltage (V)	2000
Drying T/°C	325
Drying gas (L min^−1N2)	3.00
CID gas	He
Mass range	100–500 m/z

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Electrospray ionization mass spectrometry . A 100 μL syringe, running at 18 μL h⁻¹, was connected to an electrospray ionization/ion trap mass spectrometer (Agilent HPLC-Chip/Trap XCT Ultra, Agilent Technologies, Santa Clara, CA, USA) with the nanospray interface, operating in ultra scan positive ion mode with the maximum accumulation time 300 msec. Other conditions are given in Table 1. The instrument was externally calibrated using a calibration mixture (Agilent Technologies, Santa Clara, CA, USA) containing masses of 118.09, 322.05, 622.03, 922.01, 1521.97, 2121.93 and 2721.89 m/z. The mass spectrometer was then optimized by direct infusion of 200 μg L⁻¹selenomethionine standard. All MSⁿ experiments were performed with an isolation width of 4.0 mass units and the fragmentation amplitude was 0.8 (arbitrary units). Ten parent ions were selected and isolated by the instrument, producing MS² and MS³spectra by collision induced dissociation with He gas.

High-performance liquid chromatography . Chromatographic separations were carried out with an Agilent 1100 liquid chromatograph (Agilent Technologies, Santa Clara, CA, USA) equipped with a binary HPLC pump, an autosampler, a vacuum degasser system, a temperature column compartment and a diode array detector. The outlet of the HPLC/UV detector was connected to sample inlet of the ICPMS nebulizer using 0.25 mm i.d. polyether ether ketone (PEEK) tubing of 30 cm in length. For size exclusion chromatography a Superdex 200 10/300 GL (10 mm × 300 mm, 13 μm) and a Superdex peptide 10/300 GL (10 mm × 300 mm, 13 μm) (Amersham Pharmacia Biotech, Uppsala, Sweden) were used. The column was calibrated with a mixture of 3 proteins (thyroglobulin MW = 670 kDa, ovalbumin MW = 44 kDa, myoglobin MW = 17 kDa). For ion-pairing reversed phase chromatography a Zorbax SB-C18 (4.6 mm × 150 mm, 5 μm) (Agilent Technologies, Santa Clara, CA, USA) was used. The instrumental conditions are depicted in Table 1.

Lyophilization and digestion. A Flexi-Dry MP lyophilizer (Stoneridge, NY) was used for freeze drying purposes. Freeze-dried soybean samples were digested for total selenium determination using a closed-vessel CEM Discover-Explorer microwave digestion system (CEM Corporation, Matthews, NC, USA). The computer-based microwave system, equipped with a 24-vial autosampler, is programmable for temperature, pressure (maximum 300 psi) and power (maximum 300 Watts).

Reagents and standards

All reagents were of analytical grade and no selenium was detected within the working range of these experiments. All the solutions were prepared in 18 MΩ cm⁻¹ doubly deionized water (Sybron Barnstead, Boston, MA, USA). Sodium selenite (Se(IV)), sodium selenate (Se(VI)) and heptafluorobutyric acid (HFBA) were purchased from Sigma-Aldrich, Inc. (St. Louis, MO, USA). Phenylmethanesulfonyl fluoride (PMSF), Se-Methylseleno-L-cysteine (MeSeCys) and selenocystine (SeCys₂) were purchased from Fluka (Fluka Chemie, Buchs, Switzerland). L-Selenomethionine (SeMet) and tris(hydroxymethyl)aminomethane (Tris) were obtained from Acros Organics (Morris Plains, NJ, USA). Other reagents were used: methanol (Tedia company, Inc., Fairfield, OH, USA); hydrochloric acid (Pharmco Products, Inc., Brookfield, CT, USA); nitric acid (Pharmaco, Hartford, CT, USA). The SEC standard (Bio-Rad Laboratories, Inc., Hercules, CA, USA) is a lyophilized mixture of molecular weight markers ranging from 17 000 to 670 000 Da (Fig. 2).

Procedures

Plant cultivation and supplementation. The soybean seeds were obtained from a local farmers market in Cincinnati, OH. They were transferred to individual cells after germination with doubly deionized water. Each cell contained Promix BX soil from Natorp landscape (Cincinnati, OH), which is mixture of sphagnum, peat moss, perlite, vermiculite, dolomite and calcitic lime stone. The cells were watered daily and fertilized when needed. The plants were then transplanted into troughs (1 ft × 1 ft × 6 ft). After three months of total growth from seed, the plants were supplemented with approximately 130 mg of selenium in the form of Na₂SeO₃ administered in six increments over a four week time span. Taking into account the soil volume, the plants were exposed to a soil concentration of approximately 0.9 mg kg⁻¹ during each supplementation increment. For harvesting the soybean plants were washed with doubly deionized water, separated into individual plant compartments, lyophilized, homogenized and stored at −20 °C.

Microwave oven nitric acid digestion. Lyophilized samples were treated with 1 mL of 50% HNO₃ and subjected to microwave digestion in septum sealed glass tubes. The digestion process was performed at 150 °C for 2 min by maintaining a pressure below 250 psi. The clear solution obtained was filtered, diluted to 10 mL and analyzed by ICPMS for total Se.

Extraction of Se species from soybean plants. Ground samples were treated with 2 ml of 30 mM Tris-HCl (pH 7.5) solution^31–33 containing 2% sodium dodecyl sulfate (SDS) and 0.1 mM PMSF.³⁴ (SDS was used to help solubilize proteins while PMSF was used to inhibit proteases by reacting with serine residues, so proteins containing Se would not be decomposed.³⁵) The mixture was stirred for 2 h, subsequently centrifuged at 4 °C, and 5000 rpm for 15 min using a Sorvall centrifuge (Kenro Laboratory Products, Newtown, CT, USA). The supernatant was filtered through a 0.22 μm polyvinylidene fluoride (PVDF) filter (Alltech, Deerfield, USA). Half of the filtrate was diluted 5 times and analyzed for total Se concentration, and the remaining filtrate was distributed into two vials, one of which was injected onto the SEC column and the other was further filtered through a 5 kDa cutoff membrane (Agilent Technologies, Santa Clara, CA, USA) before it was submitted for IPRP-HPLC analysis.

Fraction collection. The effluent of the IPRP-HPLC column was collected for eight injections at time intervals that represented the elution of the unidentified selenium-containing compounds. After collection, the samples were preconcentrated by evaporating with a gentle nitrogen stream³⁶ and further diluted in 50% MeOH. All collected extracts were then reanalyzed by IPRP-HPLC-ICPMS to confirm that the correct peaks were collected. The fractions were then introduced to ESI-ITMS by direct infusion.

Results and discussion

Total selenium concentrations

All the plant compartments (bean, pod, leaf and root) from microwave digestion were analyzed separately for total selenium concentrations, and the results are given in Fig. 1. The highest concentration of selenium was found in the root (151 ± 14 mg kg⁻¹, n = 3), which had direct contact with selenium supplemented soil; these results are in agreement with other Se-enriched plants.³⁷ Among the three aerial compartments, the bean (75 ± 5 mg kg⁻¹, n = 3) accumulates significantly higher concentration of selenium than the leaf (36 ± 3 mg kg⁻¹, n = 3) and pod (16 ± 2 mg kg⁻¹, n = 3). No visual plant damage was observed during the Se-enriched soybean growth, in agreement with Yang et al.²⁶ In fact, appropriate selenium supplementation promotes growth and acts as antioxidant by inhibiting lipid peroxidation and cell membrane injury.³⁸ The relatively high amount of selenium in the bean may add additional nutritional benefits to soybean, since selenium is an essential micronutrient¹ and it has been cited as a cancer chemopreventive.³⁹ Producing nutritious foods is the ultimate goal of modern agriculture. Past efforts have focused on increasing crop yields, while enhancing the concentrations of mineral micronutrients like selenium is a task of some urgency, because about half of the world’s population suffers from iron, zinc, and selenium malnutrition.¹¹ Se-enriched food is important, but the particular selenium species ingested may also be important.⁴⁰ In spite of the total selenium concentration levels, the nutritional value of selenium in the soybean also depends on the selenium species present, some indicated by the following experiments.

Fig. 1 Total selenium concentration in each compartment of the Se-enriched soybean plant. (Standard deviations for n = 3 are shown.)

Fig. 2 The SEC-ICPMSchromatogram of each compartment of the Se-enriched soybean plant. Calibration of the column was performed with a UV detector using a gel filtration standard mixture (thyroglobulin MW = 670 kDa, ovalbumin MW = 44 kDa, myoglobin MW = 17 kDa).

High molecular weight selenospecies

Size-exclusion chromatography coupled to ICPMS detection has been used to study the distribution of selenium in covalently bound high molecular weight biomolecules.^31,32,41 The Tris-HCl extract from the soybean bean was injected into a size-exclusion column (Superdex Peptide (10/300 GL)), which separates in the molecular weight range from 100 to 7000 Da, and the resulting chromatogram (not shown) displayed a large peak at void volume with an area taking ca. 82% of the total peak areas. Although this chromatogram indicated the majority of selenium in the bean was incorporated into high molecular weight (HMW) selenospecies, the resolution from the separation was poor. Another size-exclusion column, with the molecular weight range from 10 kDa to 600 kDa, was used to achieve greater resolution for the separation, and the chromatograms obtained for the various plant compartments are shown in Fig. 2a. The multiple peaks with molecular weight greater than 17 kDa indicate various HMW selenospecies extending to >600 kDa. The first peak, and most prominent, shows the majority of selenium in the bean as incorporated into large molecules. The total selenium measurement indicates the bean accumulates the majority of selenium in the aerial portions of the plant, and the SEC analysis shows >80% of the selenium in the bean is composed of HMW selenium. Selenium compounds have been tested for health benefits⁴² and chemopreventive activities,⁴³ but the effect of HMW selenium species remains unknown, because typically, selenomethionine enriched yeast, or selenomethionine alone, or with other excipients is generally taken. HMW selenium usually contains selenoamino acids such as SeMet or SeCys,^24,25 so it is interesting to compare HMW selenium with free selenoamino acids. The selenium in HMW selenium species associated with other moieties, may offer higher delivery efficiency and correspondingly less toxicity.

It was reported that SeMet was found in enzymatic digest of bean proteins of the soybean plant,^24,25 suggesting a likely presence of selenium-containing proteins. Using SEC-ICPMS a number of publications have reported the presence of high molecular weight (HMW) selenospecies in Se-enriched plants, indicating the selenium compounds supplemented in soil, usually inorganic selenium species, may be utilized by plants for HMW selenospecies synthesis.^31,32,44,45 However, none of naturally occurring HMW selenospecies have been identified in higher plants. (Lobinski et al. identified selenium-containing proteins in selenized yeast³⁵). With the substantial amount of HMW selenium, protein or other identification is possible. The identities of the HMW selenium may resolve the question of selenium essentiality, and contribute to the better understanding of the selenium metabolic pathways in plants⁹ and the additional implications for better Se nutrition.

The other three compartments of the soybean (Fig. 2b–d) contain predominantly low molecular weight selenium species eluting at 30 min, with a very small portion of HMW selenium. Since their prominent peaks elute at the same retention time, it is difficult to resolve their differences with SEC alone. To this end, the following ion-pairing reversed phase HPLC (IPRP-HPLC analysis provides further investigation of the selenium species.

Low molecular weight selenium compounds

The extract obtained by Tris-HCl solution was a mixture of water soluble moieties. If the whole extract is introduced to a Zorbax SB-C18 column, the chromatography is poor causing low mass balance as well as poor reproducibility. For example, the majority of selenium in HMW molecules does not elute from the C18 column. Therefore, a 5000 Da cutoff membrane was used. The filtrate from each compartment was injected onto the C18 column with the chromatographic conditions are given in Table 1.

Four commercially available selenium compounds—inorganic Se (a mixture of sodium selenite and sodium selenate), selenocystine (SeCys₂), Se-methylselenocysteine (MeSeCys) and selenomethionine (SeMet)—were used as standards, and were efficiently separated in 10 min, which is a 20 min reduction in analysis time from our previous study.²⁷ Better peak resolution also was achieved by using a lower concentration of HFBA and MeOH in mobile phase (Table 1). By retention-time matching the four selenium standards were found in the soybean plants and, in general, they were the major low molecular weight (LMW) selenium species (Fig. 3). SeCys₂ and SeMet are the primary selenium compounds in the bean, with about 74% of the total peak area. It has been commonly reported that SeCys₂ found in plant is usually from oxidation of selenocysteine (SeCys)⁴⁶ because SeCys is readily oxidized once exposed to air. It is possible to suggest that SeCys was the actual selenium metabolite, which then oxidized to SeCys₂ during sample handling. Further, it is also possible the SeCys came from the enzymatic digestion of selenoprotein. Both of SeCys and SeMet are the selenoamino acids incorporated into selenoproteins and selenium-containing proteins (most likely through sulfur metabolic pathways). The high content of these two amino acids is in agreement with the high content of the HMW selenium molecules, which might include one or both of these amino acids. Proposed selenium metabolic pathways for plants have shown that there may be two pathways, one of which goes to MeSeCys by methylating SeCys (thereby, minimizing formation of HMW selenium), the other of which goes to selenoprotein or selenium-containing protein through SeCys or SeMet, respectively.^9,40 The first pathway is only favored under genetic modification via a methyl transferase.^36,47 The bean produces less MeSeCys, covering only 9% of the total peak area. The bean, as a likely producer of selenium-containing proteins, appears to promote production of SeCys and SeMet, while SeCysmethylation is not a primary factor. Generally the bean contains the lowest amount of inorganic Se compared to the other three compartments, showing its high efficiency in transforming selenite to more bioavailable organoselenium forms that can be readily used for HMW selenospecies synthesis. The low content of inorganic Se also reduces the bean’s toxicity, in reference to the high level of acceptable supplementation range to be used as a dietary source.

Fig. 3 IPRP-HPLC-ICPMSchromatograms of each compartment of the Se-enriched soybean plant. (A, inorganic Se; B, SeCys₂; C, MeSeCys; D, SeMet.)

The other three compartments-pod, leaf and root share three selenium compounds, inorganic selenium, SeCys₂ and a unknown peak eluting between SeCys₂ and MeSeCys (Fig. 3). Inorganic selenium is the prominent peak for each of these compartments, and SeCys₂ is the second most abundant peak. Other selenium compounds, including MeSeCys, SeMet and other unknowns, are minor peaks occupying very small portions of the total peak areas. In the root and leaf, inorganic selenium utilizes over 90% of the peak areas, and only a very small portion of selenium is converted to organoselenium compounds.

Molecular selenium identification with ESI-ITMS. Structural characterization was carried out using ESI-ITMS, confirming the identities of IPRP-HPLC peaks. The elution “D” at 8.1 min in the bean (Fig. 3), the prominent peak in this compartment, was collected five times with the plasma off after the appropriate collection time range was established by ICPMS, and then preconcentrated by introducing pressurized nitrogen gas to accelerate evaporation. The gas also protects the fraction from oxidation, because SeMet is readily oxidized to selenomethionine oxide (C₅H₁₁NO₃Se, MW = 213.0 for ⁸⁰Se).⁴⁸ The preconcentrated fraction also contained SeMet, as seen by comparing its MS, MS² and MS³ with the corresponding spectra of standard SeMet using ESI-ITMS. (Fig. 4) In full scan (m/z 100–500), the protonated molecule [M + H]⁺ at m/z 197.8 (⁸⁰Se) was observed in both elution “D” and SeMet. In elution “D” the isotopic pattern, compared with the standard was disturbed, probably due to its presence in a complicated matrix. The molecular ion was isolated by ion trap, and helium gas was introduced into the trap to induce dissociation by collision. In elution “D” the fragments produced from m/z 197.8 showed the most intensive fragment ion was m/z 180.6 [M + H–NH₃]⁺ in the MS²spectrum. This MS² ion was isolated and further fragmented, giving the MS³spectrum for fragmentation of m/z 180.6, producing fragment ions resulting from loss of H₂O, m/z 162.7, loss of CO, m/z 152.6, breaking the C–C bond, m/z 134.6 [CH₃CH₂SeCH₂CH₂]⁺, m/z 122.5 [CH₃SeCH₂CH₂]⁺, and m/z 108.6 [CH₃SeCH₂]⁺. In these spectra no selenomethionine oxide ions were observed indicating the nitrogen gas blow down provided effective protection against oxidation.

Fig. 4 ESI-ITMSspectra of the IPRP-HPLC elution “D” showed in Fig. 3a (a) and a standard compound SeMet (b).

Conclusion

A comprehensive selenium profile was depicted for each compartment of the Se-enriched soybean plant. The bean was the most interesting one, not only because it took the majority of selenium in the aerial portions of the plant, but it also produced a very high amount of high molecular weight (HMW) selenospecies (82%). The soybean plant was shown to be very efficient in converting selenite (most likely oxidized to selenate in the soil for a better uptake) to HMW selenium molecules, which may add additional nutritional value to soybean. Soybean’s capability to produce proteins indicates that investigating the abundant HMW selenospecies in the bean should be a fertile next step in identifying specific selenoproteins or selenium-containing proteins in land plants. Further, this investigation and future endeavors will expand the understanding of selenium’s role in plants. The other three compartments: pod, leaf and root accumulated smaller selenium compounds for the most part, among which inorganic Se is the primary selenium species. The main LMW selenium species found in the bean were selenomethionine and selenocystine (oxidation product of selenocysteine). Selenomethionine and selenocysteine are usually considered as building blocks for selenium-containing proteins. The combined data of high and low molecular weight selenium species indicates the effective transformation from LMW to HMW selenospecies in the bean.

Acknowledgements

The authors would like to acknowledge support from Agilent Technologies and CEM Corporation for their instrumentation and continuing support of our programs. We would also like to thank Pam Bishop, former University of Cincinnati greenhouse manager, for her invaluable assistance in growing the plants.

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