Date Published: February 2, 2018
Publisher: Public Library of Science
Author(s): Patrick Schaub, Marta Rodriguez-Franco, Christopher Ian Cazzonelli, Daniel Álvarez, Florian Wüst, Ralf Welsch, Changjie Xu.
The net amounts of carotenoids accumulating in plant tissues are determined by the rates of biosynthesis and degradation. While biosynthesis is rate-limited by the activity of PHYTOENE SYNTHASE (PSY), carotenoid losses are caused by catabolic enzymatic and non-enzymatic degradation. We established a system based on non-green Arabidopsis callus which allowed investigating major determinants for high steady-state levels of β-carotene. Wild-type callus development was characterized by strong carotenoid degradation which was only marginally caused by the activity of carotenoid cleavage oxygenases. In contrast, carotenoid degradation occurred mostly non-enzymatically and selectively affected carotenoids in a molecule-dependent manner. Using carotenogenic pathway mutants, we found that linear carotenes such as phytoene, phytofluene and pro-lycopene resisted degradation and accumulated while β-carotene was highly susceptible towards degradation. Moderately increased pathway activity through PSY overexpression was compensated by degradation revealing no net increase in β-carotene. However, higher pathway activities outcompeted carotenoid degradation and efficiently increased steady-state β-carotene amounts to up to 500 μg g-1 dry mass. Furthermore, we identified oxidative β-carotene degradation products which correlated with pathway activities, yielding β-apocarotenals of different chain length and various apocarotene-dialdehydes. The latter included methylglyoxal and glyoxal as putative oxidative end products suggesting a potential recovery of carotenoid-derived carbon for primary metabolic pathways. Moreover, we investigated the site of β-carotene sequestration by co-localization experiments which revealed that β-carotene accumulated as intra-plastid crystals which was confirmed by electron microscopy with carotenoid-accumulating roots. The results are discussed in the context of using the non-green calli carotenoid assay system for approaches targeting high steady-state β-carotene levels prior to their application in crops.
Carotenoids are plastid-synthesized lipophilic isoprenoids which serve as precursors for pharmacologically relevant metabolites, such as crocetin or β-cryptoxanthin [1–3]. Moreover, animals including humans rely on carotenoid uptake through their diet to exploit their function as radical scavenger and provitamin A . Accordingly, there are numerous approaches to increase the carotenoid content of various crops to fight vitamin A deficiencies . Most provitamin A-rich edible tissues, such as fruits and seeds, are non-green, which restricts the use of Arabidopsis as a model system for basic research. In this study we established an Arabidopsis-derived, non-green callus assay to develop strategies for increasing and stabilizing β-carotene which can also be applied for carotenoid-derived phytochemicals. Serving as a model, the cell culture system allows evaluating suitable gene combinations to be tested prior to engineering biofortified crops.