A blackish-flowered cultivar of Catharanthus roseus accumulates high concentrations of a novel anthocyanin with a unique feature of aggregation in weak acid solutions

Abstract A cultivar of Catharanthus roseus, ‘Jams ‘N Jellies Blackberry’, was investigated for its rare blackish flower color by comparing several factors affecting flower color with another ten C. roseus cultivars that had red to purple flower colors. Petals of only ‘Jams ‘N Jellies Blackberry’ accumulated only one novel anthocyanin, peonidin 3-robinobioside (Pn 3-rob), whereas those of all the other cultivars contained multiple anthocyanins. Moreover, although ‘Jams ‘N Jellies Blackberry’ accumulated only one anthocyanin, Pn 3-rob, the total anthocyanin concentration was 2.5-fold higher in ‘Jams ‘N Jellies Blackberry’ than in the cultivar with the second-highest anthocyanin concentration ‘Equator Deep Apricot’, which had orange–red flowers. Microscopic observation of transverse sections of petals revealed that only ‘Jams ‘N Jellies Blackberry’ exhibited aggregation of anthocyanins, namely anthocyanic vacuolar inclusions (AVIs), in the vacuoles of petal epidermal cells. Furthermore, the pH of petals of ‘Jams ‘N Jellies Blackberry’ was approximately 6.0, which was significantly higher than that of all the other cultivars examined, which were in the range 5.0–5.6. Using purified Pn 3-rob and three other anthocyanins, 7-methylcyanidin 3-robinobioside, rosinidin 3-robinobioside, and hirstidin 3-robinobioside, which were the main anthocyanins accumulated in cultivars other than ‘Jams ‘N Jellies Blackberry’, the color of their solutions was measured in vitro at pH values of 3.0, 5.0, and 6.0. Pn 3-rob did not exhibit the darkest color (lowest C* and L*) at all pH conditions investigated. However, at pH 5.0 and 6.0, Pn 3-rob recrystallized immediately after dissolution, and the solution retained the dark color even though slight discoloration occurred. The feature of aggregation of anthocyanins in the solution was almost identical to that in the AVIs observed in petal epidermal cells of ‘Jams ‘N Jellies Blackberry’. The color of the anthocyanin solution was darker at pH 6.0 than at pH 5.0 for all types of anthocyanin tested. Based on these results, we could postulate that multiple factors, such as the type and high concentration of anthocyanins, high pH of the petals, and formation of AVIs, could be involved in the development of the rare blackish flower color of ‘Jams ‘N Jellies Blackberry’. Further studies should be conducted to reveal the degree of contribution of each factor to blackish flower color using progenies of ‘Jams ‘N Jellies Blackberry’ with various phenotypes and genotypes.