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John R. Stommel* and Robert J. Griesbach

Anthocyanins contribute to color development in economically important vegetables, fruits and floral crops. Their expression is critical to product sensory quality attributes, potential nutritive value, and stress response. Anthocyanins are synthesized in response to numerous environmental factors including temperature and light stress and pathogen attack. We have developed several Capsicum lines, including `02C27', expressing anthocyanin pigmentation differentially in various tissues (leaf, stem, fruit and flower). HPLC analysis demonstrated that the anthocyanins within the fruit, flower and leaves of Capsicum `02C27' were identical and that the major anthocyanidin was a delphinidin glycoside. Line `02C27' exhibits anthocyanin foliar pigmentation that is accumulated differentially in response to temperature stress. Under unfavorable low temperature (20 °C day/18 °C night), mature Capsicum leaves contained 4.6 times less anthocyanin per gram fresh weight than under high (30 °C day/28 °C; day/night) temperatures. Besides containing less anthocyanin in mature leaves, young immature leaves did not develop color as quickly under the lower temperature. Utilizing cloned and sequenced gene fragments of pepper chalcone synthase (CHS), dihydroflavonol 4-reductase (DFR), and anthocyanidin synthase (ANS), we evaluated the role of transcription in regulation of flavonol biosynthesis. Analysis of anthocyanin composition and gene expression data indicated that the block in anthocyanin formation in less pigmented leaves occurred at anthocyanin synthase. In contrast to wild tupe plants, this mutant also exhibited reduced flowering and failed to set fruit under high temperature, long day conditions.

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R.J. Griesbach

An in vivo system was developed to determine the effects of pH on naturally occurring pigment complexes within cells. The in vivo system was based on a transposable element activator (Ac) inserted into the Ph6 gene. The transposable element activator (Ac) was crossed into two genetically marked Petunia hybrida lines expressing known flavonoid pigments. Plants expressing the transposable element activator (Ac) produced variegated flowers in which the background tissue was lighter in intensity than the sectors. Depending on the genetic background in which the transposable element is expressed, progeny with darker sectors that were also redder in color than the background tissue could also be obtained. The anthocyanin and copigment composition was the same for both of the differently colored sectors and background tissue, while the pH was lower by 0.4 unit in the redder sectors. It was suggested that the Ph6 gene might be a regulatory gene that controls the expression of the pH and anthocyanin concentration.

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Roger G. Fuentes-Granados and Mark P. Widrlechner

This study was conducted to determine the inheritance of anthocyanin production and of malate dehydrogenase banding patterns in Agastache rugosa. Results of the study support the hypothesis that anthocyanin production is controlled by a single dominant gene, designated as A, for anthocyanin production. The Mdh-3 banding patterns are controlled by two alleles, each of which associated with a two-banded phenotype. A monomeric quaternary structure of MDH, which is rather atypical among plant species, can be inferred from the results. No linkage was found between the loci governing anthocyanin production and Mdh-3 banding patterns. This is the first report of heritable variability in A. rugosa.

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Aparna Gazula, Matthew Kleinhenz, Peter Ling and Joseph Scheerens

Leaf samples collected from field plots of nine lettuce varieties established in the Early (ES) and Late (LS) Summer of 2002 and 2003 in Celeryville, Ohio, were subjected to spectrophotometric measurement of anthocyanin concentrations and/or color analysis based on colorimeter and spectroradiometer readings and human panelist ratings. Interactions between year (Y), planting date (PD), and variety (V) main effects for anthocyanin concentration were significant due to shifts in response magnitude, but not direction. Anthocyanin levels were higher following LS than ES planting, regardless of Y and V, and PD effects were pronounced in 2002, when differences in average daily temperature between ES and LS plantings tended to be larger. Also, regardless of Y and PD, anthocyanin levels followed the pattern `Impuls' > `OOC 1441' > `Valeria' > `OOC1426' > `Lotto' > `SVR 9634' `OOC 1434' > `OOC 1310' > `Cireo'. Treatment-based color differences were also evident in colorimeter and spectroradiometer readings. And, panelists differentiated field-grown samples based on red color intensity. Strong correlations between analytical and instrumented and human panelist-based measures suggest that instrumented assessments of red color intensity may serve as reliable proxies for direct measures of anthocyanin levels or human panelist ratings, particularly if the aim is to establish color differences between major experimental groups and assign a quantitative, repeatable value to red color intensity.

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Satoru Kondo, Kentaro Hiraoka, Shozo Kobayashi, Chikako Honda and Norihiko Terahara

Cyanidin 3-galactoside was the primary anthocyanin in red `Tsugaru' apples [Malus sylvestris (L.) Mill. var. domestica (Borkh.) Mansf.]. The concentration of cyanidin 3-galactoside in the skin decreased from 20 to 62 days after full bloom (DAFB), then increased rapidly after 104 DAFB. Small amounts of cyanidin 3-arabinoside and cyanidin 3-glucoside were detected at 122 and 133 DAFB (harvest). The expression of five anthocyanin biosynthetic genes of chalcone synthase (MdCHS), flavanone 3-hydroxylase (MdF3H), dihydroflavonol 4-reductase (pDFR), anthocyanidin synthase (MdANS), and UDP glucose-flavonoid 3-O-glucosyltransferase (pUFGluT) was examined in the skin of red and nonred apples. In general, the expression of anthocyanin biosynthetic genes in red apples was strong in juvenile and ripening stages. The expression of MdCHS, MdF3H, pDFR, and MdANS was observed before ripening stage when anthocyanin was not detected. In contrast, the expression of pUFGluT was detected in the development stage only when anthocyanin was detected. However, the expression of all five genes was observed at 20 DAFB in fruit bagged after fertilization, and anthocyanin was not detected. The expression of MdCHS, MdF3H, pDFR, and MdANS, excluding pUFGluT, was detected at 98 DAFB in fruit bagged after 30 DAFB, and anthocyanin was not detected. These results suggest that pUFGluT may be closely related to the anthocyanin expression in apple skin at the ripening stage.

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W. Kalt, C. Lawand and C.F. Forney

Highbush blueberry (Vaccinium corymbosum) fruit of the cultivars `Bergitta', `Bluegold', and `Nelson' were harvested at six stages of maturity and evaluated for their antioxidant capacity and anthocyanin and phenolic content. Fruit of the four earliest maturities were also stored at 20 °C for up to 8 days. At the time of harvest, fruit of different maturities had substantial differences in their anthocyanin content, and less marked differences in phenolic content and antioxidant capacity. Substantial anthocyanin synthesis occurred in under-ripe fruit during 20 °C storage, and varied depending on fruit maturity at harvest. Total phenolic content changed very little during storage, and there was no change in fruit antioxidant capacity. The results suggest that anthocyanin phenolics are formed on or off the plant, primarily from other pre-existing phenolic components. Whether phenolics are present as anthocyanins or other colorless forms, has relatively little impact on antioxidant capacity.

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Md. Shahidul Islam, M. Jalaluddin, James O. Garner, M. Yoshimoto and O. Yamakawa

Sweetpotato leaves contain biologically active anthocyanins that have significant medicinal value for certain human diseases and may also be used as natural food colorants. Foliar anthocyanins and their relative abundance were investigated in leaves of sweetpotato cultivars `Shimon-1', `Kyushu-119', and `Elegant Summer' grown under artificial shading and different temperature conditions. High-performance liquid chromatography profiles of the cultivars tested showed similar peaks but with peak areas differing with cultivar, temperature and shading. The relative quantity of individual anthocyanin was YGM (Yamagawamurashaki)-1a> YGM-4b> YGM-1b> YGM-5a> YGM-0d> YGM-0a> YGM-2> YGM-0c> YGM-3> YGM-6> YGM-5b> YGM-0b> YGM-0f> YGM-0e> YGM-0g. Seven were peonidin and eight cyanidin derivatives. The highest anthocyanin contents were found in plants grown at a moderate temperature (20 °C) with lower levels at 25 and 30 °C. The leaves of plants grown in full sun accumulated significantly more total as well as the major individual anthocyanins than plants grown in 40% and 80% shade. The results indicate that growing sweetpotatoes at moderate temperatures and without shading facilitates the accumulation of anthocyanins in the leaves. The anthocyanin composition of the leaves is discussed relative to their physiological function in human health.

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Willy Kalt and Dominique Dufour

Blueberries (Vaccinium sp.) have a long history of use in native and folk medicine in North America and Europe. Today the European blueberry (bilberry) is used in a variety of pharmaceutical and food supplement products that are recommended for treating blood vessel disorders and ophthalmological conditions. Anthocyanins, the pigments that impart the blue color to blueberries, are considered the active ingredient in bilberry health products, although other related flavonoids are biomedically useful. Vaccinium flavonoids are antioxidants and are also recognized for their anticarcinogenic properties and usefulness in treating urinary tract infections. The most immediate, and perhaps greatest, opportunity for a health market for North American blueberries may be in promoting blueberries as a healthy food. As researchers continue to explore the biomedical usefulness of blueberries, the blueberry food industry should strive to retain the healthful phytochemical in their products.

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Satoru Kondo and Mako Kawai

Free and conjugated abscisic acid (ABA), anthocyanin and sugar concentrations were investigated in the skin of seedless `Pione' grapes (Vitis spp) treated with gibberellic acid (GA) and seeded `Pione' grapes. Seeded fruit were firmer than seedless fruit, until 53 days after full bloom (DAFB). The firmness of seeded fruit decreased dramatically between 43 and 53 DAFB. Anthocyanin concentrations increased in both types of fruit after 53 DAFB, but the concentration in seeded fruit was higher than in seedless fruit. Sugar concentration in the skin was higher in seedless than in seeded fruit prior to 53 DAFB, but at 80 DAFB sugar concentration was higher in the seeded fruit. ABA (free and conjugated forms) concentration in the skin showed a general increase towards harvest (80 DAFB). Skin ABA was higher in seeded than seedless fruit. After cis, trans-ABA (s-ABA) in the skin reached a maximum on 62 or 71 DAFB, its level decreased in both seeded and seedless fruit. The levels of trans, trans-ABA (t-ABA) and conjugated forms in both types of fruit failed to increase or increased only marginally after 62 or 71 DAFB. Free and conjugated ABA in the seed increased with DAFB until harvest. These results suggest that although maturation was promoted initially in the GA-treated seedless fruit, after 53 DAFB it was slowed relative to the seeded fruit. Enhanced maturation during the later stages of development of seeded fruit could be attributed to an increase in ABA concentration in the skin and the seed. These results also demonstrate that s-ABA is not metabolized in the skin to t-ABA and conjugated forms.

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Zhiguo Ju

Three years of experiments were carried out with both Delicious fruit on trees and fruit skin discs. There were two peaks of phenylalanine ammonia lyase (PAL) activity during fruit development. One occurred in the fruitlet stage and the other in the fruit enlargement stage. The first peak was coincident with anthocyanin synthesis in fruitlet but the second peak did not correlate with pigment formation during maturation. In fact, PAL activity decreased gradually during fruit maturation and coloration. Treatment with L-α-aminooxy-B-phenylpropionic acid, a specific PAL inhibitor, decreased PAL activity in fruit and in skin discs 57% and 80%, respectively, but did not change anthocyanin content. Cycloheximide inhibited anthocyanin synthesis by 76% in fruit and 85% in skin discs, but did not significantly inhibit PAL activity. On the other hand, PAL activity was positively correlated with concentrations of simple phenols which were direct products of PAL and precursors for synthesis of lignin, anthocyanin and other flavonoid.