Figure 1.
Schematic diagram of carotenoid synthesis in plants.
(A) The carotenoid biosynthetic pathway in plants. Enzymatic conversions are shown by arrows with the enzymes involved in each reaction. GGPPS: Geranyl-Geranyl Pyrophosphate Synthase; PSY: Phytoene Synthase; PDS: Phytoene Desaturase; ζ-ISO: ζ-carotene Isomerase; ZDS: ζ-carotene Desaturase; CRTISO: Carotenoid Isomerase; Z-ISO: ζ-carotene isomerase, LCYB: Lycopene β-cyclase; LCYE: Lycopene ε-cyclase; B-CHX: β-carotene Hydroxylases; E-CHX: ε-carotene Hydroxylases; ZEP: ZeaxanthinEpoxidase; VDE: Violaxanthin de-epoxidase; NSY: Neoxanthin synthase, CCD: Carotenoid cleavage dioxygenase; NCED: 9-cis-epoxycarotenoid dioxygenase. (B) Lycopene β-cyclase Reaction. The LCYB enzyme transforms one molecule of lycopene into one molecule of β-carotene or α-carotene (together with LCYE) using NADPH as cofactor.
Table 1.
Gene-specific primers used for functional characterization of DcLcyb1.
Figure 2.
Comparative alignment and sub-cellular localization of DcLCYB1.
(A) The alignment was created using ClustalW. Numbers on the right denote the number of amino acid residues. The amino acid residues which are identical in all sequences are shown in white text on a black background, whereas different residues are shown in black text on a white background. Characteristic regions of plant β-LCYs are indicated above the DcLCYB1 sequence: Conserved β-LCY region, Di-nucleotide binding site, Cyclase motifs (CM) I and II, Charged region and β-LCY motif. Domains described as essential for β-LCY activity are shown as β-LCY CAD (Catalytic Activity Domain). A plant LCY specific motif is also highlighted. SlLCYB1: Solanum lycopersicum lycopene β-cyclase 1; CaLCYB1: Capsicum annuum lycopene β-cyclase 1. (B) Subcellular localization of DcLCYB1. Leaves of two-month old tobacco plants were agroinfiltrated with A. tumefaciens carrying (a) pCAMBIA 35S::GFP, (b) pMDC85-LCYB1 and (c) recA::YFP (positive control). After 4 days, epidermal peels were observed by epi-fluorescence microscope. (a) pCAMBIA 35S::GFP – a cytoplasmic localization of soluble GFP is visible. (b) pMDC85-LCYB1 - the punctuate fluorescence is indicative of a chloroplastic localization of DcLCYB1-GFP. (c) pBI-recA- the punctuate fluorescence is indicative of a chloroplastic localization of recA::YFP. (d, e, f) Bright field images of a, b, c, respectively. All images were taken with 40x augmentation and fluorescence was observed after excitation at 489 nm.
Figure 3.
Expression levels of DcLcyb1 in leaves and storage roots during D. carota development.
Expression analysis was carried out in 4, 8 and 12 week-old plants. Different letters indicate significant differences between developmental stages and between leaves and roots. Asterisks indicate significant differences between both organs at the same stage of development. Ubiquitin was used as a normalizer, while data of leaves of 4 week-old plants were used as calibrator. A non-paired one and two tailed t-test (p<0.05) was performed.
Figure 4.
Reverse phase HPLC analysis of carotenoids accumulated in E. coli BL21 strain complemented with DcLcyb1.
Carotenoids were extracted from liquid bacterial BL21 cultures harboring pDS1BΔcrtY and transformed with either pET-Blue1 (upper pannel) or pET-Blue1/Lcyb1 (lower pannel). The bacterial pellet from each transformed strains after complementation are shown in boxes in each chromatogram. Chromatograms show that both lycopene and β-carotene are present in the strain transformed with DcLcyb1, while the control was not able to restore the mutation of crtY gene in the strain, producing only lycopene. The spectra of lycopene and β-carotene are shown in the right-hand side of the figure with numbers 1 and 2, respectively. Peak 1 corresponds to lycopene, which presents a retention time of 13 minutes and peak 2 corresponds to β-carotene with a retention time of 24 minutes [72].
Figure 5.
Over expression of DcLcyb1 increases carotenoid and β-carotene levels and expression of key carotenogenic genes.
Relative expression of DcLcyb1 gene in L6, L8 and L9 from (A) leaves and (B) storage roots of transgenic carrot plants. Ubiquitin was used as normalizer in qRT measurements. Carotenoids of D. carota L6, L8 and L9 transgenic lines were extracted from (C) leaves and (D) storage roots and carotenoid composition was determined by spectrophotometry and High Performance Liquid Chromatography. Relative expression of DcPsy1, DcPsy2 and DcLcyb2 in transgenic carrot lines from (E) leaves and (F) storage roots. Ubiquitin was used as normalizer in qRT measurements. For gene expression and carotenoid analysis three months-old plants were used. Columns and bars represent the means and SE (n = 3). Asterisks indicate significant differences between transgenic lines and the wild type plant. Non-paired one and two tailed t-tests (p<0.05) were performed for all the transgenic lines and the wild type plant.
Figure 6.
Chlorophyll content in over expresser and silenced DcLcyb1 lines of carrot plants.
Chlorophyll amount in leaves of over expresser and silenced lines was determined by spectrophotometry and HPLC. The chlorophyll amount in wild-type was set at 100%. For this analysis, leaves of three month-old plants were used. Columns and bars represent the means and SE (n = 3). Asterisks indicate significant differences between transgenic lines and the wild type plant. Non-paired one and two tailed t-tests (p<0.05) were performed for all the transgenic lines and the wild type plant.
Figure 7.
DcLcyb1 silenced lines have diminished carotenoid and β-carotene levels in leaves and roots.
DcLcyb1 transcript levels in (A) leaves and (B) storage roots in carrot transgenic lines were evaluated by qRT. Ubiquitin was used as normalizer. Carotenoid quantification in (C) leaves and in (D) storage roots of wild-type (WT) and silenced lines was determined by spectrophotometry and HPLC at 474 nm. (E) Phytoene in storage roots of wild-type (WT) and silenced lines was quantified by HPLC at 285 nm. For gene expression and carotenoid analysis, three months-old plants were used. Columns and bars represent the means and SE (n = 3). Asterisks indicate significant differences between transgenic lines and the wild type plant. Non-paired one and two tailed t-test (p<0.05) were performed for all the transgenic lines and the wild type plant.
Figure 8.
Expression of DcPsy1, DcPsy2 and DcLcyb2 decreases in transgenic carrots with reduced DcLcyb1 expression levels.
Relative expression of DcPsy1, DcPsy2 and DcLcyb2 in storage roots from six months-old AS23, AS37 and AS48 transgenic lines with reduced levels of DcLcyb1 expression were evaluated by qRT. Ubiquitin was used as normalizer. Columns and bars represent the means and SE (n = 3). Asterisks indicate significant differences between transgenic lines and the wild type plant. Non-paired one and two tailed t-test (p<0.05) were performed for all the transgenic lines and the wild type plant.