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

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

Metabolic Engineering of Potato Carotenoid Content through Tuber-Specific Overexpression of a Bacterial Mini-Pathway

BACKGROUND: Since the creation of “Golden Rice”, biofortification of plant-derived foods is a promising strategy for the alleviation of nutritional deficiencies. Potato is the most important staple food for mankind after the cereals rice, wheat and maize, and is extremely poor in provitamin A carotenoids. METHODOLOGY: We transformed potato with a mini-pathway of bacterial origin, driving the synthesis of beta-carotene (Provitamin A) from geranylgeranyl diphosphate. Three genes, encoding phytoene synthase (CrtB), phytoene desaturase (CrtI) and lycopene beta-cyclase (CrtY) from Erwinia, under tuber-specific or constitutive promoter control, were used. 86 independent transgenic lines, containing six different promoter/gene combinations, were produced and analyzed. Extensive regulatory effects on the expression of endogenous genes for carotenoid biosynthesis are observed in transgenic lines. Constitutive expression of the CrtY and/or CrtI genes interferes with the establishment of transgenosis and with the accumulation of leaf carotenoids. Expression of all three genes, under tuber-specific promoter control, results in tubers with a deep yellow (“golden”) phenotype without any adverse leaf phenotypes. In these tubers, carotenoids increase approx. 20-fold, to 114 mcg/g dry weight and beta-carotene 3600-fold, to 47 mcg/g dry weight. CONCLUSIONS: This is the highest carotenoid and beta-carotene content reported for biofortified potato as well as for any of the four major staple foods (the next best event being “Golden Rice 2”, with 31 mcg/g dry weight beta-carotene). Assuming a beta-carotene to retinol conversion of 6∶1, this is sufficient to provide 50% of the Recommended Daily Allowance of Vitamin A with 250 gms (fresh weight) of “golden” potatoes

Transformation frequencies

<p>The % of leaf discs giving at least 1 regenerant after 8 weeks on kanamycin is shown in the second column. The % of PCR-positive shoots containing the transgene are shown in the third column. The % transgenosis (fourth column) indicates the % of leaf disks giving at least 1 PCR-positive regenerant.</p

HPLC analysis of tuber and leaf pigments (µg/g dry weight)

<p>Carotenoid composition was measured via diode array HPLC (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000350#s3" target="_blank">Methods</a>) on a minimum of 8 different tubers or leaves from 4 different plants, belonging to 2 different harvests. Fold variation with respect to the wild-type is reported for each carotenoid compound and for each line.</p

Endogenous carotenoid gene expression.

<p>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. A: tubers. B: leaves. For each construct, two lines with significant carotenoid changes and one “non expressor” line (NE) are shown. The histograms show the average and SE (error bars) of determinations from at least 4 different tubers (or leaves) from 2 different plants. For details see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000350#s3" target="_blank">Materials and Methods</a>.</p

Strategy for the enhancement of the carotenoid content of potato tubers.

<p>A: Biosynthetic pathway catalyzed by the CrtB-I-Y genes. B: Schematic representation of the constructs utilized for the transformation experiments. TP: RbcS transit peptide. <i>Nos</i> and <i>Ocs</i>: Nopaline synthase and Octopine synthase polyadenylation sequences; <i>35S</i>: Constitutive CaMV <i>35S</i> promoter; <i>Pat1</i> and <i>Pat2</i>: Tuber-specific patatin promoters. For details, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000350#s3" target="_blank">Materials and Methods</a>.</p

Spectrophotometric quantitation of tuber and leaf carotenoids in transgenic lines.

<p>A: Lines transformed with the pK constructs (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000350#pone-0000350-g001" target="_blank">Figure 1B</a>). B: Lines transformed with the pP constructs (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000350#pone-0000350-g001" target="_blank">Figure 1B</a>). Data are the average of 4 independent tubers from 2 independent plants. Lines submitted to HPLC and Real Time RT-PCR analysis (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000350#pone-0000350-t002" target="_blank">Tables 2</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000350#pone-0000350-t003" target="_blank">3</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000350#pone-0000350-g004" target="_blank">Figure 4</a>) are indicated by arrows.</p

Tuber and leaf phenotypes of transgenic lines.

<p>A.Tuber phenotypes. B.Leaf phenotypes, viewed in transmitted light. The difference in size of the middle leaf is not representative.</p