Characterization of anthocyanic vacuolar inclusions in Vitis vinifera L. cell suspension cultures

Abstract

Anthocyanic vacuolar inclusions (AVIs) are intra-vacuolar structures capable of concentrating anthocyanins and are present in over 50 of the highest anthocyanin-accumulating plant species. Presence of AVIs alters pigment intensity, total anthocyanin levels, pigment hue and causes bathochromic shifts in a spatio-temporal manner within various flowers, vegetables and fruits. A year-long study on Vitis vinifera cell suspension cultures found a strong correlation between AVI prevalence and anthocyanin content, but not the number of pigmented cells, growth rate or stilbene content. Furthermore, enhancement of the prevalence of AVIs and anthocyanins was achieved by treatment of V. vinifera cell suspension cultures with sucrose, jasmonic acid and white light. A unique autofluorescence of anthocyanins was used to demonstrate microscopically that AVIs proceed from the cytosol across the tonoplast and were able to coalesce intravacuolarly, with fewer, larger AVIs predominating as cells mature. Purification and characterisation of these bodies were performed, showing that they were dense, highly organic structures, with a lipid component indicative of membrane-encasement. These purified AVIs were also shown to comprise long-chain tannins and possessed an increased affinity for binding acylated anthocyanins, though no unique protein component was detected.

Anthocyanin Vacuolar Inclusions Form by a Microautophagy Mechanism

“…The vesicular transport model postulates that anthocyanins enter the ER lumen and are transported in vesicles and/or membrane-bound organelles to the vacuole. This hypothesis is based on the observation of flavonoid-filled ERderived vesicles in Brassica napus tapetum cells (Hsieh and Huang, 2007), cytoplasmic anthocyanin-filled vesicles in grapevine (Vitis vinifera) (Conn et al, 2010;Gomez et al, 2011), and the accumulation of anthocyanins in ER bodies in Arabidopsis epidermal cells (Poustka et al, 2007). This model also contemplates the scenario that anthocyanin-containing ER domains could be engulfed by autophagosomes and delivered to the vacuole (Pourcel et al, 2010).…”

“…In sweet potato (Ipomea batatas), carnation (Dianthus caryophyllus), and lisianthus (Eustoma grandiorum), AVIs appear to lack surrounding membranes and instead consist of a protein matrix or thread-like structures (Small and Pecket, 1982;Nozue et al, 1993;Markham et al, 2000;Zhang et al, 2006;Conn et al, 2010). Besides anthocyanins, AVIs have been reported to contain a metalloprotease called VP24 in sweet potato (Nozue et al, 1997(Nozue et al, , 2003Xu et al, 2001) and tonoplast membrane lipids in grapevine cultured cells (Conn et al, 2010). In grapevine cell cultures, AVIs are enriched in acylated anthocyanins (Conn et al, 2003), suggesting that AVIs may sequester specific anthocyanin species.…”

“…The biogenesis of AVIs and whether they share the same trafficking pathway with the soluble anthocyanins is also controversial. In grapevine and lisianthus, cytoplasmic vesicles or prevacuolar compartments have been reported to enter the main vacuole and undergo intravacuolar fusion to generate AVIs (Conn et al, 2010).…”

Grape Berry Vacuole: A Complex and Heterogeneous Membrane System Specialized in the Accumulation of Solutes

“…Some enzymes involved in anthocyanin biosynthesis may be tonoplast-bound (Fritsch and Griesbach 1975). Spherical pigmented inclusions are present in the vacuoles of grape cells (Conn et al 2003(Conn et al , 2010. Sequestration of anthocyanins by anthocyanic vacuolar inclusions, loosely termed "anthocyanoplasts," is believed to increase their stability and to reduce inhibition of certain vacuolar enzymes (Conn et al 2003(Conn et al , 2010.…”

“…Spherical pigmented inclusions are present in the vacuoles of grape cells (Conn et al 2003(Conn et al , 2010. Sequestration of anthocyanins by anthocyanic vacuolar inclusions, loosely termed "anthocyanoplasts," is believed to increase their stability and to reduce inhibition of certain vacuolar enzymes (Conn et al 2003(Conn et al , 2010. Anthocyanoplasts start as vesicles in the cytosol and appear membrane-bound (Pecket andSmall 1980, Nozzolillo andIshikura 1988).…”