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Aparna Gazula*, Matthew D. Kleinhenz, Joseph C. Scheerens, Peter P. Ling and John G. Streeter

In addition to their physiological and metabolic roles, anthocyanin (Antho) levels in lettuce contribute to visual and nutritional value-based assessments of crop quality. Although 7 genes are now thought to help regulate Antho synthesis, deposition and/or degradation in lettuce, the genetic and abiotic controls of Antho levels remain less well characterized in lettuce than other plants. Previous greenhouse studies demonstrated that Antho levels in diverse lettuce varieties are a function of temperature and lighting regimen. Here, three strongly related Lolla Rossa-type varieties (`Lotto', `Valeria', and `Impuls') varying in the number of genes controlling intensity of anthocyanins were subjected to differential temperature conditions in growth chambers to better discern the independent and interactive effects of temperature (T) and variety (V) on Antho levels. Fifteen day-old seedlings were placed into one of three chambers maintained at 20 °C day/night (D/N), 30 °C/20 °C D/N or 30 °C D/N. Antho levels were measured in leaf tissue collected 30 d after transplanting. The entire experiment was replicated twice. Although significant, the T x V interaction resulted from differences in the magnitude, not direction, of the change in Antho concentrations among varieties with changes in T. This suggests that T was a main driver of Antho levels in this study. Regardless of V, Antho concentrations were highest, moderate and lowest after growth at 20 °C D/N, 30 °C/20 °C D/N and 30 °C D/N, respectively. Likewise, regardless of T, Antho levels followed the pattern `Impuls' (three genes) > `Valeria' (two genes) > `Lotto' (one gene). Correlations among instrumented and human eye-based evaluations of color are also being tested in samples from both studies.

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Shawn A. Mehlenbacher and Maxine M. Thompson

A chlorophyll deficiency expressed as yellowing of leaves was observed in hazelnut (Corylus avellana L.) progenies. Segregation ratios approximated 3 green: 1 yellow, indicating control by a single recessive gene designated chlorophyll deficient #1, for which the symbol c, is proposed. `Barcelona', `Butler', `Compton', `Lansing', Willamette', and the ornamental selection `Redleaf #3' are heterozygous. Pedigree analysis strongly suggests that all heteroxygotes inherited the recessive allele from `Barcelona'. A cross of `Barcelona' with the yellow-leafed ornamental Corylus avellana L. var. aurea Kirchn. produced no yellow-leafed seedlings, indicating that the chlorophyll deficiencies from these two sources are controlled by different loci. Progenies segregating simultaneously for this trait and the gene controlling presence of anthocyanin indicated that the two traits are inherited independently. Seedlings deficient in chlorophyll but with anthocyanin were able to survive under field conditions, while leaves of yellow-leafed seedlings lacking anthocyanin became scorched and the trees died.

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I. Iglesias, J. Graell, G. Echeverría and M. Vendrell

The influence of supplemental sprinkler irrigation on fruit color of `Oregon Spur Delicious' (Trumdor) apples (Malu×domestica Borkh.) was evaluated in the area of Lleida (NE Spain) over a 3-year period. Cooling irrigation was applied for 2 hours daily for 25-30 days preceding the harvest. Three treatments were evaluated: 1) control without overtree sprinkler irrigation; 2) sprinkler irrigation applied at midday; and 3) sprinkler irrigation applied at sunset. Fruit color was significantly affected by the cooling irrigation and also by the weather of the particular year. Increased red color and higher anthocyanin content resulted from sprinkler irrigation, especially when applied at sunset. At harvest, anthocyanin content was correlated with a*/b* and hue angle, suggesting that the colorimeter measurements could provide a nondestructive estimate of anthocyanin content.

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David L. Ehret, Brenda Frey, Tom Forge, Tom Helmer and David R. Bryla

irrigation and water management, on blueberry fruit quality attributes such as berry size, firmness, and shelf life remain largely undefined. Blueberries contain high levels of anthocyanins and other flavonoids, which show anticarcinogenic properties and are

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Hallie G. Dodson, J.B. Murphy and T.E. Morelock

Anthocyanins are naturally occurring plant pigments that are classified as flavonoids. Anthocyanins have important antioxidant properties which may help in prevention of cancer, arthritis, and cardiovascular disease. Finding common sources and possibly increasing levels of anthocyanins in food could be important to human health. This research project determined amount and type of anthocyanins in 16 cultivars and breeding lines of cowpeas (Vigna unguiculata). The information obtained from this research project will be used to improve anthocyanin content of cowpeas by breeding. Of the 16 cultivars and breeding lines, only a black cowpea breeding line, 95-356, contained measurable levels of 3 types of anthocyanins: delphinidin, peonidin, and an unknown anthocyanin. Total anthocyanin content was 0.00242 mg·g–1, which is equal to 0.21 mg per ½-cup serving. In another study with 95-356 Rabi A. Musah, found a total anthocyanin content of 121.26 mg per serving and also found three other types of anthocyanins. The difference in the studies could be explained by the storage time of two weeks after the anthocyanins were extracted, but before they were eluded in this study. Additional studies are needed to determine if cowpeas can provide anthocyanin levels comparable to other fruits and vegetables.

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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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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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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.