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

Transgene expression in leaves and tubers

<p>Values are normalized with respect to the β-tubulin transcript. For each construct, two lines with significant carotenoid changes and one “non expressor” line (NE) are shown.</p

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

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

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

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

Schematic representation of metabolite and gene expression changes in “golden” tubers.

<p>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 lines P-YBI 17 and 30 - is represented by different color hues (see legend). Asterisks mark Provitamin A carotenoids (α- and β-carotene).</p

Electron transfer reactions catalyzed by CRTI.

<p>A, Potentiometric measurement of oxygen consumption during phytoene desaturation. B, Phytoene desaturation (lycopene formation) using quinones as electron acceptors. The assays were run under an N₂ atmosphere for 30 minutes otherwise maintaining the standard conditions. The quinones used were menaquinone (−80 mV), phylloquinone (−70 mV), menadione (0 mV), duroquinone (+5 mV), Q10 (+65 mV), naphtoquinone (+70 mV) dichlophenolindophenol (+217 mV) and benzoquinone (+280 mV) all at a concentration of 240 µM. Open squares, naphtoquinones, filled symbols, benzoquinones.</p

Structure of CRTI.

<p>The crystal structure of CRTI (A) is shown in comparison with protoporphyrinogen IX oxidoreductase from <i>Myxococcus xanthus</i> (B; Protein Data Bank 2IVD). Pseudodomains are colored in blue (substrate-binding), orange (non-conserved ‘helical’ or ‘membrane binding) and green (FAD-binding). Image was generated with PyMOL. The non-ordered regions in CRTI are indicated by the numbering of adjacent residues.</p