Investigation of the in vivo organization of anthocyanins using resonance raman microspectrometry

Resonance Raman (RR) microspectrometry constitutes a new means for studying the organization of anthocyanins in living tissues. RR spectra of the pigments present in a single cell have been recorded from the skins of the mature berries of the ‘Pinot noir’ grape, as well as the petals of the common mallow. Comparison of these spectra with those obtained from model solutions of anthocyanins permits us to conclude that in the ‘Pinot noir’ berries, malvidin 3-glucoside is the main pigment. Furthermore, inside the skin it is essentially in the quinonoidal base form, whereas in the outer face of the skin it is mainly in the flavylium form. In the upper epidermis of petals of the common mallow, only malvidin 3,5-diglucoside could be detected, entirely in the cationic flavylium form. Since self-association or co-pigmentation processes do not seem to contribute much to the RR spectra, we conclude that they perturb the electronic excited state of the monomeric anthocyanin chromophores much more than they modify the corresponding electronic ground state. In the case of the skins of the mature berries of the ‘Pinot noir’ grape, dark grains, which we believe to be anthocyanoplasts, have been observed. In vivo RR spectra of the dark grains have been recorded.     

Occurrence,location and development of anthocyanoplasts

Intensely pigmented organelles (anthocyanoplasts) have been found in anthocyanin-producing cells of more than 70 species representing at least 33 families of angiosperms. When fully developed these structures are typically spherical and normally only one is present in each pigmenented cell. The development of the anthocyanoplast has been studied in both light and dark-grown red cabbage seedlings and the location of the mature organelle has been shown, by the use of isolated protoplasts and vacuoles, to be within the main cell vacuole. Evidence is presented which suggests that the anthocyanoplast is membrane-bounded and that it is the site of anthocyanin biosynthesis.