Abstract
Carotenoids of staple food crops have a high nutritional value as provitamin A components in the daily diet. To increase the levels of carotenoids, inhibition of carotenoid-cleavage dioxygenases (CCDs), which degrade carotenoids, has been considered as a promising target in crop biotechnology. In this study, suppression of the OsCCD1, OsCCD4a, and OsCCD4b genes using RNAi was verified in transgenic rice plants by quantitative RT-PCR and small RNA detection. Leaf carotenoids were significantly increased overall in OsCCD4a-RNAi lines of the T1 generation, and the highest accumulation of 1.3-fold relative to non-transgenic plants was found in a line of the T2 generation. The effects on seed carotenoids were determined via cross-fertilization between β-carotene-producing transgenic rice and one of two independent homozygous lines of OsCCD1-RNAi, OsCCD4a-RNAi, or OsCCD4b-RNAi. This showed that carotenoids were increased to a maximum of 1.4- and 1.6-fold in OsCCD1-RNAi and OsCCD4a-RNAi, respectively, with a different preference toward α-ring and β-ring carotenoids; levels could not be established in OsCCD4b-RNAi. In addition, the contents of four carotenoids decreased when OsCCD1, OsCCD4a, and OsCCD4b were overexpressed in E. coli strains accumulating phytoene, lycopene, β-carotene, and zeaxanthin. OsCCD1 and OsCCD4a had a similar high carotenoid degrading activity, followed by OsCCD4b without substrate specificity. Overall, our results suggest that suppresing OsCCD4a activity may have potential as a tool for enhancing the carotenoid content of seed endosperms and leaves in rice.
Highlights
Carotenoids, C40 terpenoid metabolites, play vital roles in plants as antenna pigments in photosynthesis, photo-protectants in the xanthophyll cycle, and precursors for the apocarotenoid hormones abscisic acid and strigolactone (Walter and Strack, 2011; Nisar et al, 2015)
The expression of OsCCD1, OsCCD4a, and OsCCD4b was examined during five stages of seed development at 10, 15, 20, 30, and 40 d after flowering (DAF) and at an additional stage that was seeds at 40 DAF that had been desiccated for 1 week (Fig. 1b)
Individual carotenoid-cleavage dioxygenases (CCDs) across the different types that are found in plants cleave targeted carotenoid substrates at different double-bond positions to yield diverse apocarotenoids, which function as pigments, volatiles, defense compounds, and signals (Walter and Strack, 2011; Hou et al, 2016)
Summary
Carotenoids, C40 terpenoid metabolites, play vital roles in plants as antenna pigments in photosynthesis, photo-protectants in the xanthophyll cycle, and precursors for the apocarotenoid hormones abscisic acid and strigolactone (Walter and Strack, 2011; Nisar et al, 2015). Dietary uptake of carotenoids benefits animals by providing health-enhancing. Humans require carotenoids as vitamin A precursors and antioxidants, and health problems are caused when they are deficient in the diet (Mayer et al, 2008; Nisar et al, 2015; Giuliano, 2017). Strategies to block carotenoid catabolism have increased the contents of nutritionally valuable β-carotene and zeaxanthin via gene-silencing of the downstream steps of β-carotene hydroxylase (BCH) in potato tubers and zeaxanthin epoxidase (ZEP) in orange fruit and the competing branch step of lycopene ε-cyclase (LCY-e) in potato tubers (Römer et al, 2002; Diretto et al, 2006; Pons et al, 2014).Wheat endosperm lacking carotenoids has been simultaneously engineered to produce carotenoids by ectopic expression of the bacterial phytoene synthase gene CrtB and to increase carotenoids by silencing the endogenous BCH-step gene TaHyd (Zeng et al, 2015)
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