Anthocyanins are flavonoid pigments synthesized in the cytoplasm and stored inside vacuoles. Many plant species accumulate densely packed, 3-to 10-mm diameter anthocyanin deposits called anthocyanin vacuolar inclusions (AVIs). Despite their conspicuousness and importance in organ coloration, the origin and nature of AVIs have remained controversial for decades. We analyzed AVI formation in cotyledons of different Arabidopsis thaliana genotypes grown under anthocyanin inductive conditions and in purple petals of lisianthus (Eustoma grandiorum). We found that cytoplasmic anthocyanin aggregates in close contact with the vacuolar surface are directly engulfed by the vacuolar membrane in a process reminiscent of microautophagy. The engulfed anthocyanin aggregates are surrounded by a single membrane derived from the tonoplast and eventually become free in the vacuolar lumen like an autophagic body. Neither endosomal/prevacuolar trafficking nor the autophagy ATG5 protein is involved in the formation of AVIs. In Arabidopsis, formation of AVIs is promoted by both an increase in cyanidin 3-O-glucoside derivatives and by depletion of the glutathione S-transferase TT19. We hypothesize that this novel microautophagy mechanism also mediates the transport of other flavonoid aggregates into the vacuole.

Main ConclusionDifferent abiotic stress conditions induce distinct sets of anthocyanins, indicating that anthocyanins have different biological functions, or that decoration patterns of each anthocyanin are used for unique purposes during stress.The induction of anthocyanin accumulation in vegetative tissues is often considered to be a response of plants to biotic or abiotic stress conditions. Arabidopsis thaliana (Arabidopsis) accumulates over 20 anthocyanins derived from the anthocyanidin cyanidin in an organ-specific manner during development, but the anthocyanin chemical diversity for their alleged stress protective functions remains unclear. We show here that, when grown in various abiotic stress conditions, Arabidopsis not only often accumulates significantly higher levels of total anthocyanins, but different stress conditions also favor the accumulation of different sets of anthocyanins. For example, the anthocyanin patterns of seedlings grown at pH 3.3 or in media lacking phosphate are very similar and characterized by relatively high levels of the anthocyanins A8 and A11. In contrast, anthocyanin inductive conditions (AIC) provided by high sucrose media are characterized by high accumulation of A9* and A5 relative to other stress conditions. The modifications present in each condition correlate reasonably well with the induction of the respective anthocyanin modification enzymes. Taken together, our results suggest that Arabidopsis anthocyanin profiles provide ‘fingerprints’ that reflect the stress status of the plants.Electronic supplementary materialThe online version of this article (doi:10.1007/s00425-014-2079-1) contains supplementary material, which is available to authorized users.

Anthocyanins are induced in plants in response to abiotic stresses such as drought, high salinity, excess light, and cold, where they often correlate with enhanced stress tolerance. Numerous roles have been proposed for anthocyanins induced during abiotic stresses including functioning as ROS scavengers, photoprotectants, and stress signals. We have recently found different profiles of anthocyanins in Arabidopsis (Arabidopsis thaliana) plants exposed to different abiotic stresses, suggesting that not all anthocyanins have the same function. Here, we discuss these findings in the context of other studies and show that anthocyanins induced in Arabidopsis in response to various abiotic stresses have different localizations at the organ and tissue levels. These studies provide a basis to clarify the role of particular anthocyanin species during abiotic stress.Anthocyanins are plant pigments of the flavonoid subclass of phenylpropanoids characterized by a 3,5,7-trihydroxylated flavylium backbone. 1 The red-to-purple color imparted by anthocyanins to flowers, fruits, and seeds act as visual deterrents to herbivores, and attractants to pollinators and seed dispersers. Anthocyanins and other flavonoids also contribute to stress tolerance in plants. There is a growing interest in understanding the mechanisms by which anthocyanins help plants cope with abiotic stress, most importantly in the context of crop yield reduction due to global climate change. Anthocyanins are commonly induced in plant vegetative tissues in response to a number of different abiotic stresses including drought, salinity, excess light, sub-or supra-optimal temperatures, and nitrogen and phosphorous deficiency. 2-8 The proposed roles of anthocyanins during abiotic stresses include quenching of ROS, 9,10 photoprotection, 11,12 stress signaling, 13,14 and xenohormesis (i.e., the biological principle that relates bioactive compounds in environmentally stressed plants and the increase in stress resistance and survival in animals that feed from them). 15,16 Plants as a group produce hundreds of structurally distinct anthocyanin species. Arabidopsis (Arabidopsis thaliana) alone produces more than 20 different types of anthocyanins, but whether they have specific functions is unknown. Whereas all anthocyanins could have identical roles, the high metabolic cost of adding numerous decorations (e.g. sugar and acyl groups) to the flavylium backbone in the different anthocyanin species makes this scenario very unlikely.We recently reported that distinct profiles of anthocyanins are induced in seedlings of Arabidopsis in response to different abiotic stresses. 4 We analyzed seedlings grown in 8 abiotic stress conditions, including high salinity, cold, and an artificial stress medium termed anthocyanin induction condition (AIC), which consists of 3% sucrose and no additional nutrients. The fact that distinct profiles of anthocyanins are induced by different abiotic stresses suggested that different anthocyanins, or profiles of anthocyanins, have different function...

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