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Gordon J. Lightbourn, John R. Stommel and Robert J. Griesbach

Anthocyanin pigmentation in leaves, flowers, and fruit imparts violet to black color and enhances both ornamental and culinary appeal. Shades of violet to black pigmentation in Capsicum annuum L. are attributed to anthocyanin accumulation. Anthocyanin production is markedly influenced by numerous environmental factors, including temperature and light stress. The objective of this study was to determine the genetic basis for differences in C. annuum anthocyanin content in response to varying environments. Growth experiments conducted under controlled environment conditions demonstrated that anthocyanin concentration was significantly higher in mature leaves in comparison with immature leaves under high light (435 μmol·s−1·m−2) conditions. High (30 °C day/25 °C night) versus low (20 °C day/15 °C night) temperature had no significant effect on anthocyanin concentration regardless of leaf maturity stage. Foliar anthocyanin concentration in plants grown under short days (10 h) with low light intensity (215 μmol·s−1·m−2) was significantly less than under long days (16 h) with low light. Under high light intensity, daylength had no effect on anthocyanin content. Three structural genes [chalcone synthase (Chs), dihydroflavonol reductase (Dfr), anthocyanin synthase (Ans)] and three regulatory genes (Myc, MybA, Wd40) were selected for comparison under inductive and noninductive environmental conditions for anthocyanin accumulation. Expression of Chs, Dfr, and Ans was significantly higher in mature leaves in comparison with younger leaves. Consistent with anthocyanin concentration, temperature had no effect on structural gene expression, whereas light positively influenced expression. Under low light conditions, temperature had no effect on Myc, MybA, and Wd40 expression; whereas under high light conditions, temperature only had an effect on MybA expression. The study of anthocyanin leaf pigmentation in C. annuum under inductive and noninductive environments provides a new approach for elucidating the molecular genetic basis of epistatic gene interactions and the resulting phenotypic plasticity.

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Gordon J. Lightbourn, Robert J. Griesbach and John R. Stommel

Color observed in plants is due to several pigments, in particular chlorophylls, carotenoids, flavonoids, and betalains. The many hues can be attributed to a number of biochemical factors, inclusive of pigment concentration, pigment combinations and their ratios, and vacuolar pH. Shades of violet to black pigmentation in pepper (Capsicum annuum L.) are attributed to anthocyanin accumulation. The color of unripe pepper fruit varies from green and yellow to ivory, through varying shades of violet and purple to nearly black. Whereas pepper fruit color is important for culinary product quality, foliar pigmentation is also an important aspect of ornamental variety appeal. Foliage and stem color may vary from green to varying shades of green/purple to nearly black. HPLC analysis of violet and black pepper fruit revealed a single anthocyanidin that was identified as delphinidin. Black fruit contained five-fold higher chlorophyll concentrations in comparison to violet fruit, which contained relatively little chlorophyll. Differences in fruit pH were not statistically significant. Similar to fruit, black pepper leaf tissue contained delphinidin as the predominant anthocyanidin, but in higher concentration relative to that found in fruit. The results demonstrate that high concentrations of delphinidin in combination with chlorophyll account for black pigmentation. Real-time PCR analysis of tissues that varied in pigmentation intensity due to varying anthocyanin concentration revealed functional, but differentially expressed, structural genes in the anthocyanin biosynthetic pathway. Analysis of regulatory gene expression identified a MYB transcription factor that was differentially expressed in response to varying anthocyanin concentration.

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John R. Stommel, Gordon J. Lightbourn, Brenda S. Winkel and Robert J. Griesbach

Anthocyanin structural gene transcription requires the expression of at least one member of each of three transcription factor families: MYC, MYB, and WD40. These transcription factors form a complex that binds to structural gene promoters, thereby modulating gene expression. Capsicum annuum L. (pepper) displays a wide spectrum of tissue-specific anthocyanin pigmentation, making it a useful model for the study of anthocyanin accumulation. To determine the genetic basis for tissue-specific pigmentation, we used real-time polymerase chain reaction to evaluate the expression of anthocyanin biosynthetic (Chs, Dfr, and Ans) and regulatory (Myc, MybA, and Wd) genes in flower, fruit, and foliar tissue from pigmented and nonpigmented C. annuum genotypes. No differences were observed in expression of the Wd gene among these tissues. However, in all cases, biosynthetic gene transcript levels were significantly higher in anthocyanin-pigmented tissue than in nonpigmented tissues. MybA and Myc transcript levels were also substantially higher in anthocyanin-pigmented floral and fruit tissues. Our results demonstrate that differential expression of C. annuum MybA as well as Myc occurs coincident with anthocyanin accumulation in C. annuum flower and fruit tissues. In contrast to the situation in flowers and fruit, differential expression of MybA and Myc was not observed in foliar tissue, suggesting that different mechanisms contribute to the regulation of anthocyanin biosynthesis in different parts of the C. annuum plant. Cloning and sequencing of MybA genomic and cDNA clones revealed two introns of 249 and 441 bp between the R2R3 domains. Whereas the Myb R2R3 domains were conserved between C. annuum and Petunia ×hybrida Vilm., the sequence of the non-R2R3 domains was not conserved, with very little homology in these related Solanaceous species.