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George L. Hosfield and Clifford W. Beninger

Seed coat color in dry bean (Phaseolus vulgaris L.) is determined by the presence or absence of tannins, flavonoids, and anthocyanins. Black beans contain three main anthocyanins that are responsible for their black seed coat color: delphinidin 3-O-glucoside, petunidin 3-O-glucoside, and malvidin 3-O-glucoside. Leaching of anthocyanins occurs in many black bean genotypes during thermal processing (i.e., blanching and cooking). Black beans that lose their dark color after processing are unacceptable to the industry. Since the marketability of black beans can be adversely affected by thermal processing, an experiment was conducted to ascertain whether pigment leaching was due to qualitative or quantitative changes in anthocyanins during processing. Four black bean genotypes that showed differential leaching of color were investigated. `Harblack' retains most of its black color after processing while `Raven' loses most of its color. `Black Magic' and `Black Jack' are intermediate between `Harblack' and `Raven' in processed color. Bean samples (119 ± 1.5 g) of the four genotypes were thermally processed in 100 x 75-mm tin cans in a pilot laboratory. Seed coats were removed from the cooked beans, freeze-dried, and placed in solutions of formic 10 acid: 65 water: 25 methanol to extract anthocyanins. The extracts were analyzed by HPLC. Although all genotypes retained some color, there were no detectable anthocyanins in seed coats of the cooked beans. In a second experiment, raw beans of each genotype were boiled in distilled water for 15 minutes. All four genotypes lost color during boiling, but `Harblack' retained most of its color and had a five-fold higher concentration of the three anthocyanins than did the other genotypes. `Harblack' may retain color better than other black beans because of physical characteristics of the seed coat.

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Clifford W. Beninger, George L. Hosfield and Muraleedharan Nair

Dry bean (Phaseolus vulgaris L.) seed coat color is determined by the presence and relative amounts of phenolics, flavonoids, and anthocyanins present in the lumen of epidermal cells. Some of these chemicals may interact with proteins of the cotyledon to form complexes that render beans hard to cook and digest. Eight genetic loci control seed pigment chemistry. When all eight loci are dominant, a shiny black seed coat results, but recessive substitutions at one or more loci yield colors ranging from white, yellow, and brown to dark violet. In order to relate Mendelian genes for seed coat color to the pigments formed, we studied eight genetic stocks that had recessive substitutions at one or more color-determining loci in an otherwise all-dominant genetic background. Seed coat from each genotype was extracted exhaustively with hexane, EtOAc, MeOH, MeOH:H2O 1:1, and H2O 100%. Silica gel thin-layer chromatography (TLC) (solvent system CHCl3:MeOH 4:1) analysis of the MeOH fraction showed that one genotype had no phenolic compounds and two had only simple phenols. Once flavonol glycoside was present in relatively large amounts in four of the genotypes, but absent in genotypes with anthocyanins. Cellulose TLC (2-dimensional, Butanol:Acetic Acid:H2O 4:1:5 first dimension, 1% HCl second dimension) of the anthocyanin-containing genotypes showed that the presence of one flavonol and three anthocyanidin-3-glycosides (UV spot color and color shift with NH3). The relative importance of the seed coat chemicals in digestibility and their antioxidant will also be discussed.

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Mark A. Kelm, James A. Flore and Clifford W. Beninger

Apple seedlings (Malus domestica Borkh.) were grown under ambient (370), 700, and 1400 μmol·mol-1 CO2 regimes and artificially damaged by removal of leaf area (0%, 15%, and 30%). Increased CO2 concentration had a highly significant effect on the concentrations of sucrose, sorbitol and phloridzin, however there were no significant interactions between CO2 concentration and leaf damage. As CO2 concentration increased there was an increase in levels of sucrose and phloridzin, whereas sorbitol concentration decreased. These findings are discussed in relation to the carbon nutrient balance hypothesis as well as other hypotheses regarding the production of plant primary and secondary compounds in response to elevated levels of CO2 and mechanical damage and/or herbivory.

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Clifford W. Beninger, George L. Hosfield and Mark J. Bassett

Three dry bean (Phaseolus vulgaris L.) genotypes differing in seedcoat color, mineral brown (P C D J G B v), yellow brown (P C D J G b v), and pale greenish yellow (P C D J g b v), were analyzed phytochemically. Kaempferol 3-O-β-d-glucoside (astragalin) was isolated and identified by nuclear magnetic resonance spectroscopy from all three genotypes, and was the main flavonoid monomer present. Flavonoid polymers (condensed tannins) were detected by thin layer chromatography, but anthocyanins were not detected in the three genotypes. High pressure liquid chromatography analyses indicated that astragalin was present at similar concentrations in pale greenish yellow and mineral brown genotypes, but was significantly lower in yellow brown. Presently, we do not know the functions of the G and B color genes, although the presence of astragalin in the three genotypes studied indicates these genes do not appear to act in a qualitative manner with regard to astragalin production, but may control the amount of astragalin present. Subtle differences in color between these genotypes may be due to the amount and type of tannins which have secondarily polymerized with phenolics and flavonoid monomers.

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Clifford W. Beninger, George L. Hosfield, Mark J. Bassett and Shirley Owens

Three common bean (Phaseolus vulgaris L.) seedcoat color (or glossiness) genotypes, differing from each other by a single substitution at a seedcoat locus, were analyzed for presence and concentration of three anthocyanins: delphinidin 3-O-glucoside, petunidin 3-O-glucoside, and malvidin 3-O-glucoside. The three anthocyanins were present in Florida common bean breeding line 5-593 (P C J G B V Asp), matte black (P C J G B V asp), and dark brown violet (P C J G b V Asp), but the amounts varied greatly depending on the genotype. Dark brown violet had 19% of the total anthocyanin content when compared to 5-593, whereas matte black had amounts intermediate between the two other genotypes. The B gene acts to regulate the production of precursors of anthocyanins in the seedcoat color pathway above the level of dihydrokaempferol formation, perhaps at the chalcone synthase or chalcone isomerase steps in the biosynthetic pathway. We hypothesize that B regulates simultaneously the flavonoid (color) and isoflavonoid (resistance) pathways. The I gene for resistance to bean common mosaic virus (BCMV) is known to be linked closely to B. It is therefore hypothesized that the I gene function may be to respond to BCMV infection by dramatically increasing (over a low constituitive level) production in the 5-dehydroxy isoflavonoid pathway, which leads to synthesis of the major phytoalexin, phaseollin, for resistance to BCMV. Alternatively, the B and I genes may be allelic. The Asp gene affects seedcoat glossiness by means of a structural change to the seedcoat. We demonstrate that Asp in the recessive condition (asp/asp) changes the size and shape of the palisade cells of the seedcoat epidermis, making them significantly smaller than either 5-593 or dark brown violet. Asp, therefore, limits the amounts of anthocyanins in the seedcoat by reducing the size of palisade cells.