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The red colour of tomato (Lycopersicon esculentum) fruits is provided by the carotenoid pigment lycopene whose concentration increases dramatically during the ripening process. A single dominant gene,Del, in the tomato mutantDeltachanges the fruit colour to orange as a result of accumulation of δ-carotene at the expense of lycopene. The cDNA for lycopene ε-cyclase (CrtL-e), which converts lycopene to δ-carotene, was cloned from tomato. The primary structure of CRTL-E is 71% identical to the homologous polypeptide fromArabidopsisand 36% identical to the tomato lycopene β-cyclase, CRTL-B. TheCrtL-egene was mapped to a single locus on chromosome 12 of the tomato linkage map. This locus co-segregated with theDelgene. In the wild-type tomato, the transcript level ofCrtL-edecreases at the ‘breaker' stage of ripening to a non-detectable level in the ripe fruit. In contrast, it increases approximatley 30-fold during fruit ripening in theDeltaplants. TheDeltamutation does not affect carotenoid composition nor the mRNA level ofCrtL-ein leaves and flowers. These results strongly suggest that the mutationDelis an allele of the gene for ε-cyclase. Together with previous data, our results indicate that the primary mechanism that controls lycopene accumulation in tomato fruits is based on the differential regulation of expression of carotenoid biosynthesis genes. During fruit development, the mRNA levels for the lycopene-producing enzymes phytoene synthase (PSY) and phytoene desaturase (PDS) increase, while the mRNA levels of the genes for the lycopene β- and ε-cyclases, which convert lycopene to either β- or δ-carotene, respectively, decline and completely disappear.
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Carotenoids are C40 isoprenoids which consist of eight isoprene units. Carotenes are hydrocarbons that are either linear or cyclized at one or both ends of the molecule, and xanthophylls are formed by the introduction of various oxygen functions to carotenes ( Goodwin 1980). The most prominent chemical feature of the carotenoids is the polyene chain, consisting of 3–15 conjugated double bonds, which is responsible for the characteristic absorption spectrum and therefore the colour of the carotenoid, and for the photochemical properties of the molecule ( Britton 1995). Due to these photochemical properties, carotenoids are essential components for all photosynthetic organisms, where they participate in a number of processes ( Frank & Cogdell 1996). These include light-harvesting, resulting in energy transfer to the chlorophylls; photoprotection by quenching triplet-state chlorophyll molecules; and scavenging singlet-state chlorophyll (reviewed by Demmig-Adams & Adams 1996). Carotenoids also serve structural functions in the photosynthetic pigment–protein complexes of the reaction centres and the light-harvesting antennae, where they are bound to specific chlorophyll/carotenoid-binding proteins.
In higher plants, carotenoids fulfil an additional important purpose as colorants of flowers and fruits. In these tissues they accumulate in chromoplasts and render bright yellow, orange or red colours that attract animals which facilitate pollination and seed dispersion.
The carotenoid biosynthesis pathway takes place within the plastids (reviewed by Cunningham & Gantt 1998;Harker & Hirschberg 1998) ( Fig. 1). The first committed step is the head-to-head condensation of two geranylgeranyl diphosphate (GGDP) molecules to produce phytoene, catalysed by the enzyme phytoene synthase (PSY). Two enzymes, phytoene desaturase (PDS) and ζ-carotene desaturase (ZDS or CRTQ), introduce four double bonds that convert phytoene to lycopene via phytofluene, ζ-carotene and neurosporene. The cyclization of lycopene is an important branching point in the pathway. One route leads to β-carotene (β,β-carotene) and its derivative xanthophylls: zeaxanthin, violaxanthin and neoxanthin. The latter two are precursors for the synthesis of the hormone abscisic acid (ABA). The alternative pathway leads to carotenoids with one β and one ε end ring, such as α-carotene and lutein which is a major xanthophyll in the light-harvesting system of most higher plants.
Figure 1. Pathway of carotenoid biosynthesis in plants and algae.
Cyclization of lycopene marks a branching point of the pathway to either α- or β-carotene. Enzymes are indicated by their gene assignment symbols: aba2, zeaxanthin epoxidase;CrtL-b, lycopene β-cyclase;CrtL-e, lycopene ε-cyclase;CrtR-b, β-ring hydroxylase;CrtR-e, ε-ring hydroxylase;Pds, phytoene desaturase (crtP in cyanobacteria);Psy, phytoene synthase (crtB in cyanobacteria);Zds, ζ-carotene desaturase (crtQ in cyanobacteria). GGDP, geranylgeranyl diphosphate.
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Carotenoid formation is a highly regulated process. Concentration and composition of leaf xanthophylls are affected by light intensity ( Ruban et al. 1994 ) and the accumulation of specific carotenoids in chromoplasts of fruits and flowers is developmentally regulated ( Fraser et al. 1994 ;Giuliano et al. 1993 ). Carotenoid accumulation during fruit ripening in tomato serves as a model system to investigate the regulation of the process. In tomato, total carotenoid concentration increases between 10- and 15-fold during fruit ripening. This change is due mainly to a 500-fold increase in the concentration of lycopene ( Fraser et al. 1994 ). Accumulation of lycopene begins at the ‘breaker' stage of fruit ripening after the fruit has reached the ‘mature green' stage. Following cloning of the genes for phytoene synthase (Psy) and phytoene desaturase (Pds), it was possible to demonstrate that the mRNA levels of these genes increase significantly during the ‘breaker' stage ( Fraser et al. 1994 ;Giuliano et al. 1993 ;Pecker et al. 1992 ). The changes in the steady-state levels of mRNA of Pds have been attributed to transcriptional regulation ( Corona et al. 1996 and unpublished data). A similar increase in mRNA was found in the genes GGPPS (for GGDP synthase), Psy and Pds during fruit ripening of bell pepper ( Camara et al. 1995 ). In contrast, the mRNA of CrtL-b, which encodes lycopene β-cyclase, decreases at the ‘breaker' stage. Evidence for transcriptional up-regulation of carotenoid genes in flowers has been described for Psy, Pds and CrtL-b ( Corona et al. 1996 ;Giuliano et al. 1993 ;Pecker et al. 1996 and unpublished data).
The cDNA for lycopene ε-cyclase has been cloned from Arabidopsis thaliana ( Cunningham et al. 1996 ). This enzyme in Arabidopsis produces only a single ε-ring in the lycopene molecule to yield δ-carotene. In this paper we report on cloning of the cDNA of CrtL-e from tomato, encoding lycopene ε-cyclase. Measurements of mRNA levels of CrtL-e indicated that down-regulation of gene expression of both types of lycopene cyclase is a major mechanism that is responsible for lycopene accumulation during tomato fruit ripening. Evidence is presented that strongly suggests that the locus Del in the fruit-colour mutation Delta encodes the gene for lycopene ε-cyclase.