Principal Accomplishment

There are at least six classes of antioxidant compounds -- flavonols, isoflavones, anthocyanins, proanthocyanidins, tocopherols, and polycarboxylic acids -- present in soybeans. An increasing number of studies have documented the significant value of many classes of these compounds not only as potent antioxidants, but also as antitumor agents and cardioprotective compounds. We have discovered the molecular basis of the soybean T locus (an important region governing pigmentation in the soybean seed coat) to be a flavonoid 3' hydroxylase which influences the type of antioxidant pigments in the seed coat. The RNA for this enzyme is very abundant in the developing seed coats but does not occur at all in the cotyledons. Along with a related enzyme, the flavonoid 3 hydroxylase, we conclude that these two enzymes are critical for production of the proanthocyanidin antioxidant compounds in the pigmented seed coats whereas the absence of their messenger RNAs in the cotyledons would favor production of isoflavone compounds that are higher in abundance in the soybean seed cotyledons. Further study of these could lead to ways to manipulate production of the compounds in different plant parts by genetic engineering approaches.

Executive Summary

We undertook to evaluate the potential of soybeans as a source for commercially valuable antioxidants. Our focus was on oligomeric proanthocyanidins (OPCs), a group of phenolic compounds which have large and increasing sales as dietary supplements. The compounds are known to occur in soybean seeds, especially those of highly colored varieties, which are of limited usefulness in the traditional soy markets.

The pigments of developing seed coats were studied in the Williams Black cultivar. During maturation, the green seeds passed through an intermediate pink-to-red stage and finally a purplish-black coloration. Overproduction of the anthocyanin, cyanidin-3-O-glucoside (which is red in dilute solution) gave rise to a black appearance to the seed coat.

The Williams Black cultivar also contained significant levels of OPCs. We were able to separate oligomers of these compounds (ranging in size from two to four subunits) by high-performance liquid chromatography. We confirmed the antioxidant activity of the mixture by a kinetic assay, but lacked sufficient material to test the individual oligomers.

Genetic investigations demonstrated that the production of polyphenols in the seed coat was principally governed by two enzymes (flavonoid hydroxylases) whose synthesis was directed by the T locus of the soybean genome. The RNA which performed their synthesis was particularly abundant in the seed coats of highly pigmented varieties. The fact that the RNA was largely absent from the cotyledon, hypocotyls, and leaf tissue of the varieties tested was correlated with the absence of these compounds in cell culture. However, studies with a variety of elicitors indicated that the production of other polyphenols was strongly influenced in these materials.

Project Highlights

We collected seed coats during seed development of pigmented (proanthocyanidin containing) and non-pigmented genotypes, isolated mRNA, and conducted the initial microarray experiments with arrays containing 9216 unique cDNAS on glass slides to survey the global expression patterns of these genes in these lines. These arrays also contain a collection of genes representing the flavonoid pathway and included those repetitively throughout the arrays.

Chemical investigations have recently been directed toward the separation and tentative identification of polyphenolic antioxidants from pigmented soybean seeds. An improved liquid chromatographic separation has permitted us to isolate individual oligomers of proanthocyanidins (OPCs), the presumed most active soy antioxidants, from one another. Mass spectrometric analysis of the separated OPCs has shown that di-, tri-, and tetrameric isomers eluted successively from the column, and were well separated from other polyphenols in the extract. This technique will permit the preparation of usable quantities of the individual compounds to examine their chemical properties, including their antioxidant efficiency, and also to establish the stereochemistry of the isomers by NMR spectroscopy.

Considerable effort was applied to the separation of the proanthocyanidin material from the anthocyanins to help elucidate the nature of the "black" coloration. Epicatechin was identified as the major proanthocyanidin of the immature green seed coat in the black cultivars only (not present in the yellow cultivars); small amounts of proanthocyanidin dimers were also present at the immature stage. By observing the developing seeds in the pods of the Williams Black cultivar, we noted that as they ripen, the seeds proceeded from a green to a pinkish-red stage. The red seed coats then rapidly blackened, either in the pod or following removal from the cotyledon. Corresponding to the visible color changes, the proanthocyanidin profile shifted with much less epicatechin in the mature seed coat and the predominance of cyanidin-3-O-glucoside as the major anthocyanin; only minor amounts of two additional anthocyanin constituents were also formed. Evaluation of the separated constituents confirmed the antioxidant efficiency of the polyphenols.

Soy cell cultures were initiated from both hypocotyl and seed coats of explants from parallel yellow and black seeded lines. Rapidly generating cell cultures have been successfully produced from Williams and NKS19-10 yellow & black soybean cotyledon, hypocotyls, and leaf tissue explants. Immature seed coat tissue was specifically targeted for explanting as the gene(s) responsible for the high proanthocyanidin content in black lines are localized in the seed coats. Isoflavone profiles were quantified in both callus and suspension cultures. Callus and suspension cultures were exposed to various physical (UV radiation; low temperature stress; timing of subculture [aging]) and chemical (sucrose concentration, elicitation with naringin, naringenin, and methyl jasmonate) microenvironmental factors in order to shift phytochemical production in vitro. Elicitation (using acetate, methyl jasmonate, or UV light ) was used as a trigger to provoke differential accumulation of bioflavonoids in cell cultures of Glycine max. While proanthocyanidins were not induced to accumulate in cell cultures under any treatment, UV irradiated callus cultures of yellow-seeded soybean accumulated higher concentrations of daidzein and genistein, known UV induced isoflavonoids, than either non-irradiated cultures or black seeded soybean. Black-seeded hypocotyl suspension cultures showed decreased levels of daidzin, MGD, MGG, daidzein, and genistein when subjected to UV radiation. Methyl jasmonate similarly provoked higher bioflavonoid accumulation, in both yellow and black seeded lines.

Additional Support and Collaboration

The Illinois Soybean Program Operating Board and United Soybean Board are supporting research by Vodkin following up some of the results obtained on this project.

Lila has consulted with a natural products and phytomedicinals laboratory group at Rutgers (Raskin lab) and as a result have planned additional elicitation experiments with acetate and chitin which have proven stimulus value in their research with black bean seedlings of a different species (not soy). This collaboration has also led to an international phytomedicinal project involving scientists from Central Asian republics of the former Soviet Union.

Publications and Presentations

Zabala, G., and Vodkin, L.O. 2002. The pleitropic tawny pubescence locus (T) encodes a flavonoid 3' hydroxylase. Molecular characterization of gray pubescence mutations (t). Proc. 9th Biennial Conference on Molecular and Cellular Biology of the Soybean. Urbana, IL. No. 506.

Kopsombut, Sinee P., K. A. Marley, R. A. Larson, and M. A. L. Smith. 2002. UV and Visible Radiation Effects on Flavonoid Production in Cell Cultures of Vitis vinifera and Glycine max. 10th International Association of Plant Tissue Culture and Biotechnology Congress: Plant Biotechnology 2002 and Beyond. Orlando, FL. Abstract # 1189. In Vitro Cellular & Developmental Biology - Abstract Issue 38:69-A.

Kopsombut, Sinee P., L. S. Kull, K. A. Marley, R. A. Larson, and M. A. L. Smith. 2002. UV Radiation Effects on Flavonoid Production in Cell Cultures of Vitis vinifera and Glycine max. Proceedings of the Functional Foods for Health 11th Annual Retreat

Zabala, G.C. and Vodkin, L.O. 2003. Cloning of the pleiotropic T locus in soybean and two recessive alleles that differentially affect structure and expression of the encoded flavonoid 3' hydroxylase. Genetics 163: 295-309.

Kopsombut, S., K. A. Marley, R. A. Larson, and M. A. Lila. 2003. Differential Accumulation of Flavonoids in Cell Cultures of Soy and Grape: Implications for Pharmacological Research. Congress on In Vitro Biology, Portland, OR. Abstract #P

Zabala, G. C. and Vodkin, L. O. 2003. Characterization of mutations that inactivate the pleitropic soybean tawny pubescence locus. Plant, Animal, and Microbe Genome XI. San Diego, CA., p. 273.

Student Support

Sinee Kopsombut was supported as a graduate student (Lila, advisor) during the project. Undergraduates were trained by Vodkin's group to learn routine lab duties and DNA extractions. Undergraduates are now pursuing graduate degrees.