Dissection of tomato lycopene biosynthesis through virus-induced gene silencing

Abstract

Lycopene biosynthesis in tomato (Solanum lycopersicum) fruits has been proposed to proceed through a poly-cis pathway catalyzed by phytoene synthase (PSY), two desaturases (phytoene desaturase [PDS] and ζ-carotene desaturase [ZDS]), and two cis-trans isomerases (ζ-carotene isomerase [ZISO] and prolycopene isomerase [CrtISO]). The mechanism of action of these enzymes has been studied in Escherichia coli, but a systematic study of their in vivo function is lacking. We studied the function of nine candidate genes (PSY1, PSY2, PSY3, PDS, ZDS, ZISO, CrtISO, CrtISO-Like1, and CrtISO-Like2) using virus-induced gene silencing (VIGS) coupled to high-resolution liquid chromatography coupled with diode array detector and mass spectrometry, which allowed the identification and quantitation of 45 different carotenoid isomers, including linear xanthophylls. The data confirm the confinement of the VIGS signal to the silenced fruits and the similarity of the phenotypes of PSY1- and CrtISO-silenced fruits with those of the yellow flesh and tangerine mutants. Light was able to restore lycopene biosynthesis in ZISO-silenced fruits. Isomeric composition of fruits silenced at different metabolic steps suggested the existence of three functional units, comprising PSY1, PDS/ZISO, and ZDS/CrtISO, and responsible for the synthesis of 15-cis-phytoene, 9,9'-di-cis-ζ-carotene, and all-trans-lycopene, respectively. Silencing of a desaturase (PDS or ZDS) resulted in the induction of the isomerase in the same functional unit (ZISO or CrtISO, respectively). All-trans-ζ-carotene was detectable in nonsilenced fruits, greatly increased in ZDS-silenced ones, and disappeared in CrtISO-Like1-/CrtISO-Like2-silenced ones, suggesting the existence of a metabolic side branch, comprising this compound and initiated by the latter enzymes.

Figure 1.

Lycopene biosynthesis pathway in tomato fruits. A, Proposed pathway, showing the main compounds and enzymes involved. B, Candidate genes involved in lycopene biosynthesis in tomato (red), Arabidopsis (green), and grape (black). Each dot represents one gene. C, Expression heat map (Log2 scale) of candidate lycopene biosynthesis genes in different tissues of tomato, analyzed by Illumina RNA-Seq. MG, Mature green fruit; B, breaker fruit; B+10, ripe fruit 10 d after breaker stage. Detailed data are shown in Supplemental Table S1. [See online article for color version of this figure.]

Figure 2.

Fruit-specific VIGS of lycopene biosynthesis genes. A, Agroinjection of mature green F6DR fruits. B, Fruits from the same branch, injected with different constructs, display construct-specific phenotypes at ripening, confirming that the virus does not spread to adjacent fruits. [See online article for color version of this figure.]

Figure 3.

Visual and HPLC phenotypes of silenced fruits at B+10. The top two panels show wild-type MM fruits and F6DR transgenic fruits at B+10. The other panels show F6DR fruits injected with different silencing constructs. Representative visual phenotypes of the fruits and chromatographic profiles recorded at 450 nm are shown in each panel. Peak labeling is as follows: I to III indicate early compounds in Figure 1; IV, lycopene isomers; V, β-carotene; VI, lutein; and VII, neurosporene isomers. The online absorbance spectra of the different compounds are shown in Supplemental Figure S9. uAU, Micro-absorbance units. [See online article for color version of this figure.]

Figure 4.

Carotenoid composition of silenced and mutant fruits at B+10. A, Amounts of the different carotenoid species in wild-type MM (LA2706), F6DR, and silenced fruits (Fig. 3), plotted as stacked bars. B, Amounts of the different carotenoid species for their wild-type Ailsa Craig (AC; LA2838A), yellow flesh (r; LA3532), and tangerine (t; LA3183) fruits, plotted as stacked bars. Detailed data are shown in Supplemental Tables S4 to S6. [See online article for color version of this figure.]

Figure 5.

Light partially restores lycopene biosynthesis in ZISO-silenced, but not CrtISO-silenced, fruits. Agroinjected fruits were put in darkness (right) or left in the light (left) until B+10, and then anthocyanin-free sectors were dissected and analyzed by HPLC. A to C, Visual phenotypes. Arrows point at lycopene accumulation in peripheral tissues of light-grown, ZISO-silenced fruits. D, Carotenoid composition of samples. Data are the average of four biological replicates. Detailed data are shown in Supplemental Table S7. [See online article for color version of this figure.]

Figure 6.

Isomer composition of silenced fruits. A, Levels of carotenoid isomers in silenced fruits. Detailed data are shown in Supplemental Table S8. B, Structure of the main isomers accumulated. P1, Hydroxy phytoene; P2, 15-cis-phytoene; P3, all-trans-phytoene; Pfl1, hydroxy phytofluene; Pfl2, 9,15-di-cis-phytofluene; Pfl3 and Pfl4, unidentified phytofluene isomers; Z1, 9,15,9′-tri-cis-ζ-carotene; Z2 and Z3, unidentified ζ-carotene isomers; Z4, 9,9′-di-cis-ζ-carotene; Z5, 9-cis-ζ-carotene; Z6, all-trans-ζ-carotene; Z7, hydroxy ζ-carotene; N1, 7,9,9′-tri-cis-neurosporene; N2 to N4, unidentified neurosporene isomers; L1, 13-cis-lycopene; L2, 5,13′-di-cis-lycopene; L3, 9-cis-lycopene; L4, 5,9′-cis-lycopene; L5, hydroxy lycopene; L6 and L7, unidentified lycopene isomers; L8, all-trans-lycopene; L9, 5-cis-lycopene; Prolyc, prolycopene (7,9,9′,7′-tetra-cis-lycopene). [See online article for color version of this figure.]

Figure 7.

Gene expression in silenced tissues. Each panel shows gene expression in anthocyanin-free sectors of F6DR fruits injected with the construct indicated. Transcript levels of the ROS and DEL transgenes and of the different carotenoid genes shown in Figure 1 were measured through real-time RT-PCR and were first normalized for expression of the housekeeping α-ACTIN gene. The ROS and DEL transcript levels were normalized to those in uninfected fruits, while carotenoid transcript levels were normalized to those in TRV/DR fruits. Target genes (indicated by black arrows) show 70% to 99% silencing. Additionally, some nontarget genes show significant repression or induction (*P value < 0.05; **P value < 0.01). Data are the average ± sd of at least three biological replicates. [See online article for color version of this figure.]

Figure 8.

Proposed model for lycopene biosynthesis in tomato fruits. Three metabolic units, composed of PSY1, PDS/ZISO, and ZDS/CrtISO, catalyze the synthesis of 15-cis-phytoene, 9,9′-di-cis-ζ-carotene, and all-trans-lycopene (and of their hydroxylated derivatives). CrtISO-L1 and -L2 are active in a metabolic side branch comprising all-trans-ζ-carotene. Blue arrows indicate regulatory loops inducing specific transcripts in response to the inhibition of specific biosynthetic steps. [See online article for color version of this figure.]