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.

Plastid Localization of the Key Carotenoid Enzyme Phytoene Synthase Is Altered by Isozyme, Allelic Variation, and Activity

“…Thus, our results suggested that Zm-PSY1-NP was located in fibrillar plastoglobuli, which initiate from globular plastoglobuli in the presence of high concentrations of carotenoids. The presence of carotenoids in fibrils was supported by the use of the Spectral Dye Separation tool in LAS AF software (Leica), applied to the fluorescence intensity spectra of Zm-PSYs-GFP constructs expressed in protoplasts prepared from etiolated leaves of Zm-PSY1 knockout maize (see 168, Amino acid represented by Asn (N) in Zm-PSY1 and Ser (S) in all other PSYs; 257, amino acid represented by Thr (T) in Zm-PSY1 and Pro (P) in all other PSYs; 174 and 190, respective amino acids are shown to affect the activity of PSY in cassava and tomato (Gady et al, 2011); 285, mutation at this site inactivates SQS (Gu et al, 1998). Hyphens indicate putative cleavage site for chloroplast transit sequence, predicted by ChloroP.…”

Investigating the role of vitamin C in tomato through TILLING identification of ascorbate-deficient tomato mutants

“…As an example, stable over-expression in tomato and in strawberry of the newly discovered GDP-l-galactose phosphorylase (VTC2/VTC5) with the GDP-mannose epimerase (GME) led to two to six-fold increases in ascorbate levels (Bulley et al 2012). Recently published results also highlighted how the functional analysis of various target genes in tomato can benefit from the availability of allelic series of mutations identified in tomato EMS mutant collections by TILLING (Di Matteo et al 2013;Gady et al 2012;MacAlister et al 2012). Indeed, EMS mutagenesis is well adapted to tomato, in contrast to other gene inactivation approaches such as the T-DNA insertional mutagenesis commonly used in Arabidopsis that involves tedious and lengthy steps of plant transformation and requires large mutant populations to reach saturation mutagenesis in tomato (Emmanuel and Levy 2002).…”

Genetic integrity assessment of cryopreserved tomato(Lycopersicon esculentum Mill.) genotypes

“…As the most powerful antioxidant in the carotenoid family, lycopene plays an important role in the prevention of cancer and chronic diseases (Rao and Agarwal, 2000;Terry et al, 2008). Tomato carotenoids are beneficial not only for human health but also for major breeding traits such as fruit color (Gady et al, 2012). Therefore, we considered LCY-B gene an important candidate for genetic stability testing following cryopreservation.…”