Daucus carota DcPSY2 and DcLCYB1 as Tools for Carotenoid Metabolic Engineering to Improve the Nutritional Value of Fruits

Abstract

Carotenoids are pigments with important nutritional value in the human diet. As antioxidant molecules, they act as scavengers of free radicals enhancing immunity and preventing cancer and cardiovascular diseases. Moreover, α-carotene and β-carotene, the main carotenoids of carrots (Daucus carota) are precursors of vitamin A, whose deficiency in the diet can trigger night blindness and macular degeneration. With the aim of increasing the carotenoid content in fruit flesh, three key genes of the carotenoid pathway, phytoene synthase (DcPSY2) and lycopene cyclase (DcLCYB1) from carrots, and carotene desaturase (XdCrtI) from the yeast Xanthophyllomyces dendrorhous, were optimized for expression in apple and cloned under the Solanum chilense (tomatillo) polygalacturonase (PG) fruit specific promoter. A biotechnological platform was generated and functionally tested by subcellular localization, and single, double and triple combinations were both stably transformed in tomatoes (Solanum lycopersicum var. Microtom) and transiently transformed in Fuji apple fruit flesh (Malus domestica). We demonstrated the functionality of the S. chilense PG promoter by directing the expression of the transgenes specifically to fruits. Transgenic tomato fruits expressing DcPSY2, DcLCYB1, and DcPSY2-XdCRTI, produced 1.34, 2.0, and 1.99-fold more total carotenoids than wild-type fruits, respectively. Furthermore, transgenic tomatoes expressing DcLCYB1, DcPSY2-XdCRTI, and DcPSY2-XdCRTI-DcLCYB1 exhibited an increment in β-carotene levels of 2.5, 3.0, and 2.57-fold in comparison with wild-type fruits, respectively. Additionally, Fuji apple flesh agroinfiltrated with DcPSY2 and DcLCYB1 constructs showed a significant increase of 2.75 and 3.11-fold in total carotenoids and 5.11 and 5.84-fold in β-carotene, respectively whereas the expression of DcPSY2-XdCRTI and DcPSY2-XdCRTI-DcLCYB1 generated lower, but significant changes in the carotenoid profile of infiltrated apple flesh. The results in apple demonstrate that DcPSY2 and DcLCYB1 are suitable biotechnological genes to increase the carotenoid content in fruits of species with reduced amounts of these pigments.

Keywords: Daucus carota; DcLCYB1; DcPSY2; Malus domestica; Solanum lycopersicum; carotenoid; metabolic engineering; transgenic tomatoes.

FIGURE 1

The main steps of carotenoid biosynthesis and related pathways in plants. The methylerythritol 4-phosphate (MEP) pathway produces isoprenoids in plastids leading to the production of geranylgeranyl pyrophosphate (GGPP). Phytoene synthase (PSY) is the first enzyme in the carotenogenic pathway and produces phytoene. In plants, phytoene is then desaturated and isomerized by phytoene desaturase (PDS), ζ–carotene desaturase (ZDS), ζ–carotene isomerase (Z-ISO) and prolycopene isomerase (CRTISO) forming lycopene. In bacteria, these reactions are carried out by a single multifunctional enzyme, phytoene desaturase (CRTI). β-carotene is produced if lycopene is cyclized only by lycopene β-cyclase (LCYB) whilst α-carotene is produced if both LCYB and lycopene ε-cyclase (LCYE) are involved. β-carotene hydroxylase (CHYB) and ε-carotene hydroxylase (CHYE) produce lutein. CHYB produces zeaxanthin, which can be converted into violaxanthin by zeaxanthin epoxidase (ZEP). The reverse step can be carried out by violaxanthin de-epoxidase (VDE). Violaxanthin is the precursor of abscisic acid. Dotted arrows indicate multiple enzymatic reactions. Solid gray arrows represent different steps of the carotenogenic pathway. The blue arrow indicates consecutive desaturations carried out by bacterial CRTI.

FIGURE 2

pCP-CG constructs used for the functional assessment of DcPSY2, DcLCYB1, and XdCRTI. The pCP backbone is derived from pB7FWG2.0, with the incorporation of a specifically designed Multiple Cloning Site (MCS; see Supplementary Figure 1). Different carotenogenic genes were cloned into the MCS, under the control of the tomato polygalacturonase promoter (pPG) and the Nos terminator (NosT). (A) pPSY2. The single gene and fruit-specific expression cassette PG:DcPSY2 was chemically synthesized and cloned into pCP. (B) pLCYB1. The single gene and fruit-specific expression cassette PG:DcLCYB2 was chemically synthesized and cloned into pCP. (C) pPSY2-CRTI. A double gene and fruit-specific expression cassette including the PG promoter driving the XdCRTI gene fused to a transit peptide (PG:tpXdCRTI) was chemically synthesized and cloned into pPSY2. (D) pPSY2-CRTI-LCYB1. The single gene and fruit-specific expression cassette PG:DcLCYB2 was cloned into pPSY2-CRTI.

FIGURE 3

Subcellular localization of DcPSY2 and XdCRTI. Transient transformation of tomato fruits agroinfiltrated with DcPSY2:GFP or XdCrtI:GFP fused to a transit peptide (tp). The expression of both constructs was driven by the pPG promoter. Fluorescence was observed 4 days after transient transformation. (a) GFP fluorescence of DcPSY2:GFP. (b) Chlorophyll autofluorescence of (a). (c) Merge of panels (a,b), showing colocalization of chlorophyll and the GFP signal. (d) Bright field of (a–c). (e) GFP fluorescence of tp:XdCRTI:GFP. (f) Chlorophyll autofluorescence of (e). (g) Merge of panels (e,f), showing colocalization of chlorophyll and GFP signal. (h) Bright field of (e–g). The TRITC filter U-N31001 was used for visualizing GFP fluorescence, with an excitation wavelength between 465 and 495 nm. Emission was collected between 513 and 556 nm. Chlorophyll autofluorescence was assessed by using a U-MNG2 filter, with an excitation wavelength between 530 and 550 nm and emission collected at 590 nm. Scale bar = 50 μm.

FIGURE 4

Fruit phenotypes and expression analysis in T1 transgenic fruits. (a) Representative WT tomato fruit. (a.1) Cross-section of (a). (b) Representative tomato fruit from pPSY2 line. (b.1) Cross-section of (b). (c) Representative tomato fruit from pLCYB1 line. (c.1) Cross-section of (c). (c) Representative tomato fruit from pPSY2-CRTI line. (d.1) Cross-section of (d). (e) Tomato fruit from pPSY2-CRTI-LCYB2. (e.1) cross-section of (e). Scale bar = 1 cm. (f) q-RTPCR in fruits of pPSY2, (g) pLCYB1, (h) pPSY2-CRTI, and (i) pPSY2-CRTI-LCYB1 transgenic lines. The relative expression was normalized to that of the Actin gene of S. lycopersicum. The expression analysis was performed with three biological replicas (cDNAs) from five fruits, each measured twice.

FIGURE 5

Total and individual carotenoid quantification in pCP-CG transgenic lines. (A) pPSY2, (B) pLCYB1, (C) pPSY2-CRTI, and (D) pPSY2-CRTI-LCYB1 transgenic lines. Total carotenoids were quantified in fruits by spectrophotometry at 474 nm and individual carotenoids by HPLC expressed in μg/gDW. Asterisks indicate significant differences respect to the control (WT) determined by one-way ANOVA and Dunnet post-test, *p < 0.05, **p < 0.01, ***p < 0.001. In (D) the results were analyzed with a two-way t-test, ****p < 0.0001.