Mechanisms of Apocarotenoid Metabolism and Signaling
Plant Physiology
Final Report Abstract
Carotenoid degradation severely affects approaches to provide enhanced levels of provitamin A carotenoids in crops, such as Golden Rice endosperm and cassava roots. While enzymatic cleavage affects off-branching plant growth regulator biosynthesis which do not affect carotenoid losses severely, the major cause for carotenoid degradation is non-enzymatic oxidation into apocarotenoids. Concluded from turnover measurements as well as degradation kinetics of Golden Rice endosperm after harvest, this process occurs with a high rate, thus, in living tissues, mechanisms have to be assumed which further metabolize the carotenoid oxidation products generated, especially considering that most apocarotenoids are cytotoxic. Apart from a few reports, e.g., on glycosylation of xanthophyll-derived cleavage products, knowledge on these mechanisms is scarce. In this project, we provoked increased carotenoid and apocarotenoid degradation by increasing the accumulation of carotenoids in roots of Arabidopsis achieved by overexpressing the rate-limiting enzyme of the pathway, phytoene synthase (PSY). These roots show high levels of mainly β-carotene and derived β- apocarotenoids, assuming transcriptome changes which allow to identify gene products involved in apocarotenoid metabolization. Carotenoid pathway-specific transcriptome changes affected individual genes involved in β-carotene metabolism, suggesting feedback responses to reduce the amounts of β-carotene formed in consequence of PSY expression in transgenic roots. Similarly, concluded from enhanced transcript levels, another drain of surplus carotenoids might be towards abscisic acid (ABA) and strigolactones (SL) although steady-state hormone levels remained unchanged. Besides pathway-specific transcriptome changes we analysed potential apocarotenoid metabolizing pathways and identified differentially regulated genes known from reactive electrophilic species and xenobiotic detoxification processes. Interestingly, several gene products, such as aldehyde reductases, aldo-keto reductases, alkenal reductases and aldehyde dehydrogenases were identified and capable of converting apocarotenoid substrates into correspondingly modified products in vitro with recombinant enzymes. Moreover, additional genes which were differentially regulated, suggested secondary modification and transportation processes which show similarities to the process of crocin formation and storage, a group of glucose conjugates of the apocarotenoid crocetin dialdehyde accumulating in saffron stigma. As levels of the dipeptide glutathione (GSH) were increased in carotenoid accumulating roots, we investigated whether apocarotenoids form GSH adducts, a mechanism which is known to be involved in the detoxification of other plant compounds. However, extensive in vitro and in vivo approaches revealed that GSH adduct formation is not a major process and cannot explain the high GSH levels observed. However, transcriptome data indicated that increased GSH levels are involved in detoxifying glyoxal and methylglyoxal via the glyoxalase system. Both products are highly reactive and accumulate upon continued degradation of apocarotenoids as the shortest possible products, but are generated as by-products in several other pathways as well. Glyoxalase-mediated detoxification of glyoxal and methylglyoxal yields products which are introduced into primary pathways. Finally, a combined analysis of transcriptomic and metabolomic data suggested that this allows to recycle carotenoid-derived carbon to fuel into other pathways including the synthesis of isopentenyl diphosphate, indicating a possible carbon recycling process for carotenoid re-synthesis. The project successfully unravelled enzymatic apocarotenoid metabolizing processes and identified a possible cyclic system for carotenoid biosynthesis. The findings will allow deeper understanding of carotenoid turnover regulation and help to develop approaches to stabilize provitamin A carotenoids in future projects.
Publications
- (2020) Characterization of Cauliflower OR Mutant Variants. Front Plant Sci 10: 1716
Welsch R, Zhou X, Koschmieder J, Schlossarek T, Yuan H, Sun T, Li L
(See online at https://doi.org/10.3389/fpls.2019.01716) - (2020) Plant apocarotenoid metabolism utilizes defense mechanisms against reactive carbonyl species and xenobiotics. Plant Physiol 185: 331–351
Koschmieder J, Wüst F, Schaub P, Álvarez D, Trautmann D, Krischke M, Rustenholz C, Mano J, Mueller MJ, Bartels D, Hugueney P, Beyer P, Welsch R
(See online at https://doi.org/10.1093/plphys/kiaa033) - (2020) Quantification of Carotenoid Pathway Flux in Green and Nongreen Systems. In: Rodríguez-Concepción M, Welsch R (eds) Plant and food carotenoids: methods and protocols, 279–291
Koschmieder J, Welsch R
(See online at https://doi.org/10.1007/978-1-4939-9952-1) - (2022) Color recycling: metabolization of apocarotenoid degradation products suggests carbon regeneration via primary metabolic pathways. Plant Cell Reports 2022 1: 1–17
Koschmieder J, Alseekh S, Shabani M, Baltenweck R, Maurino VG, Palme K, Fernie AR, Hugueney P, Welsch R
(See online at https://doi.org/10.1007/s00299-022-02831-8) - (2022) Golden Rice - lessons learned for inspiring future metabolic engineering strategies and synthetic biology solutions. In: Carotenoids: Carotenoid and apocarotenoid biosynthesis, metabolic engineering and synthetic biology. Wurtzel E (Ed); Methods in Enzymology, 671. Elsevier
Welsch R, Li L
(See online at https://doi.org/10.1016/bs.mie.2022.03.014)
