Induced point mutations in the phytoene synthase 1 gene cause differences in carotenoid content during tomato fruit ripening

Abstract

In tomato, carotenoids are important with regard to major breeding traits such as fruit colour and human health. The enzyme phytoene synthase (PSY1) directs metabolic flux towards carotenoid synthesis. Through TILLING (Targeting Induced Local Lesions IN Genomes), we have identified two point mutations in the Psy1 gene. The first mutation is a knockout allele (W180*) and the second mutation leads to an amino acid substitution (P192L). Plants carrying the Psy1 knockout allele show fruit with a yellow flesh colour similar to the r, r mutant, with no further change in colour during ripening. In the line with P192L substitution, fruit remain yellow until 3 days post-breaker and eventually turn red. Metabolite profiling verified the absence of carotenoids in the W180* line and thereby confirms that PSY1 is the only enzyme introducing substrate into the carotenoid pathway in ripening fruit. More subtle effects on carotenoid accumulation were observed in the P192L line with a delay in lycopene and β-carotene accumulation clearly linked to a very slow synthesis of phytoene. The observation of lutein degradation with ripening in both lines showed that lutein and its precursors are still synthesised in ripening fruit. Gene expression analysis of key genes involved in carotenoid biosynthesis revealed that expression levels of genes in the pathway are not feedback-regulated by low levels or absence of carotenoid compounds. Furthermore, protein secondary structure modelling indicated that the P192L mutation affects PSY1 activity through misfolding, leading to the low phytoene accumulation.

Fig. 1

Simplified overview of the carotenoid pathway. Highlighted in bold are the genes up-regulated in chromoplast-containing tissues. DXS 1-deoxy-d-xylulose-5-phosphate synthase, DXR deoxyxylulose 5-phosphate reductoisomerase, HDR hydroxymethylbutenyl diphosphate reductase, IPI IPP isomerase, GGPS geranylgeranyl diphosphatesynthase, PSY phytoene synthase, PDS phytoene desaturase, ZDS z-carotene desaturase, CRTISO carotenoid cis-trans-isomerase, LCY-B lycopene β-cyclase, CYC-B β-lycopene cyclase, CRTR-B β-carotene hydroxylase, CYP97A and C P450 carotenoid β- and ε-hydroxylases, VDE violaxanthin desaturase, ZEP zeaxanthin de-epoxidase, NXS neoxantin synthase, LCY-E ε-lycopene cyclase

Fig. 2

Graphical representation of mutation positions in the Psy1 gene and wild-type and mutant proteins sequence alignment. A PARSESNP representation (Taylor and Greene 2003) of the Psy1 gene based on genomic sequence. Mutations are positioned in the third exon. Mutation inducing the W180* is represented with a red arrowhead and mutation P192L by a black arrowhead. B ClustalW protein alignment of the wild-type and the two mutant proteins. Mutation positions are highlighted in yellow. Highlighted in green is the TARGETP-predicted chloroplastic transit peptide (Giorio et al. 2008). Highlighted in grey is the Trans_IPPS_HH conserved domain (trans-isoprenyl diphosphate synthases) identified with NCBI CDD tool

Fig. 3

Control and mutant fruit phenotypes at Mature Green, Breaker + 3 and Breaker + 7 ripening stages (left–right). Control fruit come from the same line as the mutant ones and thus, share background mutations but do not carry a mutation in Psy1. A Whole and open control fruit from line 1804. B Whole and open control fruit from line 5381. C Whole and open fruit from line 1804 carrying the PSY1 P192L amino acid substitution. D Whole and open fruit from line 5381 carrying the PSY1 W180* point mutation resulting in a Psy1 knockout allele

Fig. 4

HPLC carotenoid profiling of control lines, line 1804 carrying the P192L substitution and line 5381 carrying the W180* mutation in Psy1. Carotenoids were measured from fruit at three developmental and ripening stages: Mature Green, Breaker + 3 and Breaker + 7 stages. A Fruit phytoene content expressed as area under the phytoene curve. B Fruit lycopene content expressed in μg of lycopene/mg of fruit fresh weight. C Fruit β-carotene content expressed in μg of lycopene/mg of fruit fresh weight. D Fruit lutein content expressed in μg of lycopene per mg of fruit fresh weight. Significant difference (Student’s t test) in compound content in mutant lines compared to controls are represented with one or more asterisks with the following levels of significance: *P < 0.05; **P < 0.01; ***P < 0.001

Fig. 5

Quantitative PCR-based expression analysis of genes involved in carotenoid biosynthesis during ripening in control and mutant lines. Eleven genes selected for their role in the tomato carotenoid pathway (Fig. 1) were studied at three ripening stages, Mature Green (MG), Breaker + 3 (B + 3) and Breaker + 7 (B + 7), to follow their expression levels in the two mutant lines (W180* and P192L) and in their respective controls. For each ripening and for each mutant line and non-mutant sibling, five biological repeats were sampled and analysed for gene expression. Relative expression is plotted as ΔCt values, obtained as follows: (Ct value of target gene) − (Ct value of Actin), to correct for differential cDNA concentration between samples. ΔCt values of the two control lines were averaged and shown on the graph. Statistical calculations were performed with both individual and averaged control values. Errors bars represent the variation between biological replicas. For all genes, significant difference (Student’s t test) in relative expression in mutant lines compared to controls was calculated for all lines and ripening stages. Only in line W180* was Psy1 expression significantly (P < 0.05) lower than control at B + 3 and B + 7 ripening stages

Fig. 6

PSY wild-type and P192L mutant proteins secondary structure prediction using PBIL GOR4 tool. A Secondary structure prediction of the PSY1 wild-type protein. B Secondary structure prediction of the PSY1 P192L mutant protein. An additional alpha helix is predicted to form at the mutation position (black arrowhead)