Silencing of beta-carotene hydroxylase increases total carotenoid and beta-carotene levels in potato tubers

Abstract

Background: Beta-carotene is the main dietary precursor of vitamin A. Potato tubers contain low levels of carotenoids, composed mainly of the xanthophylls lutein (in the beta-epsilon branch) and violaxanthin (in the beta-beta branch). None of these carotenoids have provitamin A activity. We have previously shown that tuber-specific silencing of the first step in the epsilon-beta branch, LCY-e, redirects metabolic flux towards beta-beta carotenoids, increases total carotenoids up to 2.5-fold and beta-carotene up to 14-fold.

Results: In this work, we silenced the non-heme beta-carotene hydroxylases CHY1 and CHY2 in the tuber. Real Time RT-PCR measurements confirmed the tuber-specific silencing of both genes . CHY silenced tubers showed more dramatic changes in carotenoid content than LCY-e silenced tubers, with beta-carotene increasing up to 38-fold and total carotenoids up to 4.5-fold. These changes were accompanied by a decrease in the immediate product of beta-carotene hydroxylation, zeaxanthin, but not of the downstream xanthophylls, viola- and neoxanthin. Changes in endogenous gene expression were extensive and partially overlapping with those of LCY-e silenced tubers: CrtISO, LCY-b and ZEP were induced in both cases, indicating that they may respond to the balance between individual carotenoid species.

Conclusion: Together with epsilon-cyclization of lycopene, beta-carotene hydroxylation is another regulatory step in potato tuber carotenogenesis. The data are consistent with a prevalent role of CHY2, which is highly expressed in tubers, in the control of this step. Combination of different engineering strategies holds good promise for the manipulation of tuber carotenoid content.

Figure 1

Endogenous carotenoid gene expression. Transcript levels were measured through Real Time RT-PCR and were first normalized for expression of the housekeeping β-tubulin gene, and then for the expression levels in the Wt. Data show the average and SE (error bars) of determinations from at least 4 different tubers (or leaves) from 2 different plants. For details see Methods.

Figure 2

Schematic representation of metabolite and gene expression changes in engineered tubers. Boxes represent the metabolic intermediates, arrows represent the genes catalyzing the various reactions. Fold induction or repression with respect to the wild-type – averaged over three transgenic lines- is represented by different hues of red or green, respectively (see legend). White means that no data are available. Asterisks indicate significance of the fold variation with respect to the Wt in an ANOVA test (*: p ≤ 0.05; **: p ≤ 0.01; ***: p ≤ 0.001). (A) AS-e lines 1,2,3 [3]. (B) AS-h lines 1,2,3 (this paper).