Metabolic engineering of potato tuber carotenoids through tuber-specific silencing of lycopene epsilon cyclase

Abstract

Background: Potato is a major staple food, and modification of its provitamin content is a possible means for alleviating nutritional deficiencies. beta-carotene is the main dietary precursor of vitamin A. Potato tubers contain low levels of carotenoids, composed mainly of the xanthophylls lutein, antheraxanthin, violaxanthin, and of xanthophyll esters. None of these carotenoids have provitamin A activity.

Results: We silenced the first dedicated step in the beta-epsilon- branch of carotenoid biosynthesis, lycopene epsilon cyclase (LCY-e), by introducing, via Agrobacterium-mediated transformation, an antisense fragment of this gene under the control of the patatin promoter. Real Time measurements confirmed the tuber-specific silencing of Lcy-e. Antisense tubers showed significant increases in beta-beta-carotenoid levels, with beta-carotene showing the maximum increase (up to 14-fold). Total carotenoids increased up to 2.5-fold. These changes were not accompanied by a decrease in lutein, suggesting that LCY-e is not rate-limiting for lutein accumulation. Tuber-specific changes in expression of several genes in the pathway were observed.

Conclusion: The data suggest that epsilon-cyclization of lycopene is a key regulatory step in potato tuber carotenogenesis. Upon tuber-specific silencing of the corresponding gene, beta-beta-carotenoid and total carotenoid levels are increased, and expression of several other genes in the pathway is modified.

Figure 1

Carotenoid biosynthesis pathway. The enzymes involved in the various reactions are indicated.

Figure 2

Construction of plasmids for Agrobacterium-mediated transformation. The patatin B33 promoter and a region of the LCY-e transcript were amplified, respectively, from genomic DNA and tuber cDNA using primers inserting appropriate restriction sites at the ends, resequenced, and cloned in the pBI101 plasmid [21]. White triangles indicate the direction of transcription, black triangles the direction of the coding strand. For details see Methods.

Figure 3

Pigmentation of Wt and transgenic tubers. Tubers were photographed immediately after harvesting.

Figure 4

Transgene expression. (A): GUS activity was measured fluorimetrically. (B): AS-e tranSgene expression was measured via Real Time RT-PCR. For details see Methods.

Figure 5

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.