Carotenoid Steady-State Regulation
Final Report Abstract
The dynamics of carotenoid homeostasis were studied in Arabidopsis lines with carotenoid steady-state perturbations introduced by the overexpression of the rate-limiting enzyme phytoene synthase (PSY). In leaves, excess PSY protein was partially inactivated by stromal localization while enzymatically active PSY was present in membranes and yielded a twofold increase in phytoene amounts compared with the wild type. However, increased pathway flux did not affect leaf carotenoid levels, but was compensated by accordingly higher levels of various glycosidically bound apocarotenoids. In addition, the pattern of these apocarotenoids changed significantly in leaves of CCD4 but remained unchanged in CCD1 mutant line. These results identify apocarotenoid glycosylation as a novel mechanism in leaf carotenoid homeostasis regulation and suggest an essential contribution of CCD4 in enzyme-mediated carotenoid breakdown. Generation and analysis of Arabidopsis callus was optimized in order to establish a fast nongreen system suitable to study carotenoid homeostasis. This revealed an overlay of massive carotenoid breakdown with a significant pathway activity as determined by norflurazoneinhibition experiments. Analysis of Arabidopsis lines differing in phytoene synthesis capacity characterized the carotenoid breakdown as a dynamic process which was able to compensate a certain threshold of pathway flux requiring higher levels for carotenoid crystal accumulation. These crystals are present within plastids as shown by co-localization of fluorescence-labelled plastid marker proteins and crystal birefringence. Analysis of ccd1 and ccd4 callus excluded a major contribution of these enzymes in carotenoid breakdown during callus formation and suggested the involvement of other enzymes or non-enzymatic processes. Volatile or glycosidically bound apocarotenoids were not detected in callus, suggesting alternative sequestration or processing mechanisms. Transcriptome analysis with crystal-accumulating Arabidopsis roots revealed large similarities with transcriptome changes induced during lipid peroxidation. This suggests that hyperaccumulating carotenoids are partially subjected to oxidative degradation and that defense mechanisms involved in apocarotenoids scavenging might share similar signaling pathways like those involved in lipid stress. Due to similar carotenoid crystal formation upon increased pathway flux in AtPSY-overrexpressing lines and cauliflower OR mutant curd, a putative involvement of OR in carotenoid homeostasis regulation was investigated. Although a high phytoene synthesis rate in (colourless) wild-type cauliflower curd was determined, it could not ultimately be determined whether higher pathway flux is the exclusive cause for carotenoid accumulation in cauliflower OR mutant curd. However, we found that PSY interacts with cauliflower and Arabidopsis OR proteins using a yeast two-hybrid split ubiquitin system. Furthermore, callus generated from Arabidopsis lines overexpressing the OR mutant gene accumulated higher carotenoid levels and correlated with increased PSY protein levels while PSY expression was unaffected. These results strongly suggest that OR is involved in PSY protein homeostasis. Reduced PSY protein turnover caused by OR mutant versions might result in higher pathway flux resulting in carotenoid increases similar to those observed upon PSY overexpression.
Publications
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Carotenoid accumulation in roots: same target - different principles. Abstracts 16th International Symposium on carotenoids, Krakow, Poland, Acta Biologica Cracoviensia Vol. 53 suppl. 1, 87, 2011
Welsch R., Arango J., Maass D., Bär C., Beyer P.
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“Arabidopsis as a tool to study carotenoid homeostasis”, German Botanical Symposium, Tübingen, Germany, September 2013
Welsch R., Hübner M., Zhou X., Li L. and Beyer P.
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“Arabidopsis as a tool to study carotenoid homeostasis”, Gordon Research Conference Carotenoids, Ventura, USA, January 2013
Welsch R., Hübner M., Wüst F., Li L. and Beyer P.
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Carotene hydroxylase activity determines the Levels of Both α-Carotene and Total Carotenoids in Orange Carrots. The Plant Cell, May 2014, tpc.113.122127
Arango J., Jourdan M., Geoffriau E., Beyer P. and Welsch R.
