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

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

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

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

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

Affinity of p19 to perfect or mismatch-containing duplex si/miRNAs <i>in vitro</i>.

<p>Band shift assay of perfect duplex siR171 (A), and mismatched miR171a (B), miR171b (C) and miR171c (D) duplex RNAs’ with p19 protein. The structure of dsRNAs is shown above the gel pictures. Direct measurement of the absolute apparent dissociation constant Kd values (F) were calculated as previously described [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005935#ppat.1005935.ref057" target="_blank">57</a>] based on the quantification of band intensities of p19:dsRNA bound fraction as a function of p19 protein concentration (E). Relative dissociation constant (Krel) was calculated by normalization of Kd values to Kd of siR171 (F).</p

p19 protein expressing transgenic <i>Nicotiana benthamiana</i> (p19syn) plants.

<p>(A) Specific developmental phenotype of p19syn plants compared to wild type plants. Three representative independent transgenic lines are shown alongside wild-type <i>N</i>. <i>benthamiana</i> plant. (B) Western blot of p19syn transgenic and wild-type plants from lines shown in (A). The presence of p19 transgene does not impact NbAGO1 or NbAGO2 protein levels. Protein loading is shown below (StainFree). (C) Stem length from cotyledons to the fourth and last leaf insertion in wt and T1 p19syn plants at 8–9 leaf stage (lines:1–29 and 1–57) were used to show the elongated internode trait of p19syn plants. Bars: standard deviation. (D) Wild type and p19syn plants agroinfiltrated with GFP and GFP+p19 constructs as indicated. Pictures were taken at 4 dpi. (E) p19-mediated inhibition of RNA silencing of tobacco magnesium protoporphyrin chelatase subunit I <i>(ChlI)</i> (a key chlorophyll biosynthetic gene) induced by CMV + Y-satRNA infection (14 dpi, left panel), silencing of <i>ChlI</i> causes yellowing on wt plants. Northern blot hybridization of CMV + Y-satRNA infected wt or p19syn plants; nucleolar small RNA U6 was used as an internal control (right panel).</p