Biosynthesis of Chlorophyll and Other Isoprenoids in the Plastid of Red Grape Berry Skins

Despite current knowledge showing that fruits like tomato and grape berries accumulate different components of the light reactions and Calvin cycle, the role of green tissues in fruits is not yet fully understood. In mature tomato fruits, chlorophylls are degraded and replaced by carotenoids through the conversion of chloroplasts in chromoplasts, while in red grape berries, chloroplasts persist at maturity and chlorophylls are masked by anthocyanins. To study isoprenoid and lipid metabolism in grape skin chloroplasts, metabolites of enriched organelle fractions were analyzed by high-performance liquid chromatography-high-resolution mass spectrometry (HPLC-HRMS) and the expression of key genes was evaluated by real-time polymerase chain reaction (PCR) in berry skins and leaves. Overall, the results indicated that chloroplasts of the grape berry skins, as with leaf chloroplasts, share conserved mechanisms of synthesis (and degradation) of important components of the photosynthetic machinery. Some of these components, such as chlorophylls and their precursors, and catabolites, carotenoids, quinones, and lipids have important roles in grape and wine sensory characteristics

Differential accumulation of pelargonidin glycosides in petals at three different developmental stages of the orange-flowered gentian (<i>Gentiana lutea</i> L. var. <i>aurantiaca</i>)

<div><p>Corolla color in <i>Gentiana lutea</i> L. exhibits a yellow/orange variation. We previously demonstrated that the orange petal color of <i>G</i>. <i>lutea</i> L. var. <i>aurantiaca</i> is predominantly caused by newly synthesized pelargonidin glycosides that confer a reddish hue to the yellow background color, derived from the carotenoids. However, the anthocyanin molecules of these pelargonidin glycosides are not yet fully identified and characterized. Here, we investigated the regulation, content and type of anthocyanins determining the petal coloration of the orange-flowered <i>G</i>. <i>lutea</i> L. var. <i>aurantiaca</i>. Anthocyanins from the petals of <i>G</i>. <i>lutea</i> L. var. <i>aurantiaca</i> were characterized and quantified by HPLC-ESI-MS/MS (High-performance liquid chromatography-electrospray ionization-tandem mass spectrometry) coupled with a diode array detector in flowers at three different stages of development (S1, S3 and S5). Eleven pelargonidin derivatives were identified in the petals of <i>G</i>. <i>lutea</i> L. var. <i>aurantiaca</i> for the first time, but quantitative and qualitative differences were observed at each developmental stage. The highest levels of these pelargonidin derivatives were reached at the fully open flower stage (S5) where all anthocyanins were detected. In contrast, not all the anthocyanins were detected at the budlet stage (S1) and mature bud stage (S3) and those corresponded to more complex pelargonidin derivatives. The major pelargonidin derivatives found at all the stages were pelargonidin 3-<i>O</i>-glucoside, pelargonidin 3,5-<i>O</i>-diglucoside and pelargonidin 3-<i>O</i>-rutinoside. Furthermore, the expression of <i>DFR</i> (<i>dihydroflavonol 4-reductase</i>), <i>ANS</i> (<i>anthocyanidin synthase</i>), <i>3GT</i> (<i>UDP-glucose</i>:<i>flavonoid 3-O-glucosyltransferase</i>), <i>5GT</i> (<i>UDP-glucose</i>:<i>flavonoid 5-O-glucosyltransferase</i>) and <i>5AT</i> (<i>anthocyanin 5-aromatic acyltransferase</i>) genes was analyzed in the petals of three developmental stages, showing that the expression level of <i>DFR</i>, <i>ANS</i> and <i>3GT</i> parallels the accumulation of the pelargonidin glucosides. Overall, this study enhances the knowledge of the biochemical basis of flower coloration in <i>Gentiana</i> species, and lays a foundation for breeding of flower color and genetic variation studies on <i>Gentiana</i> varieties.</p></div

Concentrations of provitamin A carotenoids, lutein, phytoene, phytofluene and α-tocopherol in fetal bovine serum and Caco-2 intestinal cells before and after 4h incubation with diluted mixed micelles generated during <i>in vitro</i> digestion of GP17 and GP30 boiled tubers.

<p>Concentrations of provitamin A carotenoids, lutein, phytoene, phytofluene and α-tocopherol in fetal bovine serum and Caco-2 intestinal cells before and after 4h incubation with diluted mixed micelles generated during <i>in vitro</i> digestion of GP17 and GP30 boiled tubers.</p

Contribution of a 150 g serving of boiled wild type and golden potatoes to the estimated average requirement (EAR)^* for retinol activity equivalents (RAE) and vitamin E [44].

<p>Contribution of a 150 g serving of boiled wild type and golden potatoes to the estimated average requirement (EAR)^{<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0187102#t004fn001" target="_blank">*</a>} for retinol activity equivalents (RAE) and vitamin E [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0187102#pone.0187102.ref044" target="_blank">44</a>].</p

Carotenoid and α-tocopherol contents in raw and boiled wild-type and golden potato (GP) tubers.

<p>Carotenoid and α-tocopherol contents in raw and boiled wild-type and golden potato (GP) tubers.</p

Bioaccessible provitamin A carotenoids, lutein, phytoene, phytofluene and α-tocopherol in digested potato tubers (μg/g DW).

<p>Bioaccessible provitamin A carotenoids, lutein, phytoene, phytofluene and α-tocopherol in digested potato tubers (μg/g DW).</p

Relative accumulation (%) of provitamin A carotenes, lutein, carotenoid precursors and αTC by Caco-2 cells.

<p>Data are mean ± SD; n = 6 monolayers of Caco-2 cells incubated with the mixed micelle fraction generated by simulated digestion of a pooled sample prepared from 10 tubers from 5 plants of each genotypes. Different letters above bars indicate that means differ significantly (<i>p <</i> 0.01).</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

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

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