Overexpression of the rice carotenoid cleavage dioxygenase 1 gene in Golden Rice endosperm suggests apocarotenoids as substrates in planta

Abstract

Carotenoids are converted by carotenoid cleavage dioxygenases that catalyze oxidative cleavage reactions leading to apocarotenoids. However, apocarotenoids can also be further truncated by some members of this enzyme family. The plant carotenoid cleavage dioxygenase 1 (CCD1) subfamily is known to degrade both carotenoids and apocarotenoids in vitro, leading to different volatile compounds. In this study, we investigated the impact of the rice CCD1 (OsCCD1) on the pigmentation of Golden Rice 2 (GR2), a genetically modified rice variety accumulating carotenoids in the endosperm. For this purpose, the corresponding cDNA was introduced into the rice genome under the control of an endosperm-specific promoter in sense and anti-sense orientations. Despite high expression levels of OsCCD1 in sense plants, pigment analysis revealed carotenoid levels and patterns comparable to those of GR2, pleading against carotenoids as substrates in rice endosperm. In support, similar carotenoid contents were determined in anti-sense plants. To check whether OsCCD1 overexpressed in GR2 endosperm is active, in vitro assays were performed with apocarotenoid substrates. HPLC analysis confirmed the cleavage activity of introduced OsCCD1. Our data indicate that apocarotenoids rather than carotenoids are the substrates of OsCCD1 in planta.

Mechanism behind Petal Color Mutation Induced by Heavy-Ion-Beam Irradiation of Recalcitrant Chrysanthemum Cultivar

“…In contrast, the CCD1s reported so far lack a plastidtargeting signal and is located in the cytoplasm (Bouvier et al, 2003;McCarty and Klee, 2006). In addition, major substrates of CCD1 in planta were shown to be carotenoid cleavage products (C27-apocarotenoids; Floss et al, 2008;Ilg et al, 2010). In 'Jimba', CCD1 transcript levels were not correlated with carotenoid content, indicating that CCD1 does not affect carotenoid content in chrysanthemum petals.…”

Carotenoid Biosynthesis in Arabidopsis: A Colorful Pathway

“…However, this is not always the case. For example, CCD1 and/or CCD4 levels negatively correlate with carotenoid levels in Chrysanthemum and orchid flowers (Ohmiya et al, 2006;Chiou et al, 2010), strawberry fruit (Garcia-Limones et al, 2008), maize endosperm (Vallabhaneni et al, 2010), and potato tubers (Campbell et al, 2010), but not in Ipomea flowers (Yamamizo et al, 2010), citrus fruit (Kato et al, 2006) or rice endosperm (Ilg et al, 2010). In Arabidopsis seeds, carotenoid levels might be controlled by the rate of their degradation by CCD1, since mutant seeds defective in this enzyme show increased carotenoid levels whereas transgenic seeds overexpressing CCD1 display reduced amounts of these pigments (Auldridge et al, 2006b).…”

Origins of Grape and Wine Aroma. Part 1. Chemical Components and Viticultural Impacts

“…Four subfamilies have been identified-CCD1, CCD4, CCD7, and CCD8-and cleavage may be symmetric or asymmetric depending on the enzyme and carotenoid substrate (Auldridge et al 2006, Walter et al 2010, Young et al 2012). Recently, studies in Crocus sativa, rice, and mycorrhizal roots of Medicago truncatula indicate that CCD4 and CCD7 may be localized in the plastid and the C 13 -and C 27 -apocarotenoids obtained from carotenoid cleavage are exported to the cytosol where further cleavage by CCD1 occurs, yielding C 13 -and C 14 -apocarotenoid products (Floss et al 2008, Rubio et al 2008, Ilg et al 2010). CCD7 and CCD8 are thought to be involved in formation of strigolactone, a plant hormone that inhibits shoot branching (Ruyter-Spira et al 2013).…”