Effect of gamma-Irradiation on the Biosynthesis of Carotenoids in the Tomato Fruit

Fruits of the lutescent tomato genetic line were exposed to γ-radiation at different stages of maturity to determine the effect of ionizing radiation on carotenoid synthesis in the ripening fruit. Irradiation generally resulted in the inhibition of carotenogenesis. The effect was more pronounced at the higher dosage and in less mature fruit. Lycopene synthesis was inhibited more extensively than β-carotene synthesis. The total carotenoid content was also generally lower in irradiated fruits. It was proposed that the β-carotene in the tomato fruit is formed by a pathway not involving lycopene.     

An increase of lycopene content in tomato fruit is associated with a novel Cyc-B allele isolated through TILLING technology

Tomato Cyc-B gene encodes a chromoplast-specific lycopene β-cyclase that converts lycopene to β-carotene during ripening of the fruit. By screening the tomato Red Setter mutant population with the TILLING method, we identified eight new alleles at the Cyc-B locus. Results of greenhouse phenotypic analysis revealed that the novel A949G Cyc-B allele produced modifications in the carotenoid profile and content of tomato petals and fruit. The cyc-b7 genotype, carrying the A949G Cyc-B allele, was therefore evaluated in an open field trial for standard agronomic traits as well as carotenoid content of the fruit. Results of the field trial confirmed that the induced A949G missense mutation favored the accumulation of lycopene in the fruit with no detrimental effects on the yield or on other agronomic and technological properties such as fruit firmness and Brix degree of fruit juice. On the basis of these results, it can be affirmed that the A949G Cyc-B allele constitutes a useful new genetic variant which can be used for improving carotenoid content in tomato fruit and for the development of new tomato commercial lines. Finally, the results presented here furthermore demonstrate that TILLING is a powerful methodology not only as a confirmatory system for gene functional analysis but also for selecting new gene variants useful for genetic improvement of important crops.     

Fruit-localized phytochromes regulate lycopene accumulation independently of ethylene production in tomato

We show that phytochromes modulate differentially various facets of light-induced ripening of tomato fruit (Solanum lycopersicum L.). Northern analysis demonstrated that phytochrome A mRNA in fruit accumulates 11.4-fold during ripening. Spectroradiometric measurement of pericarp tissues revealed that the red to far-red ratio increases 4-fold in pericarp tissues during ripening from the immature-green to the red-ripe stage. Brief red-light treatment of harvested mature-green fruit stimulated lycopene accumulation 2. 3-fold during fruit development. This red-light-induced lycopene accumulation was reversed by subsequent treatment with far-red light, establishing that light-induced accumulation of lycopene in tomato is regulated by fruit-localized phytochromes. Red-light and red-light/far-red-light treatments during ripening did not influence ethylene production, indicating that the biosynthesis of this ripening hormone in these tissues is not regulated by fruit-localized phytochromes. Compression analysis of fruit treated with red light or red/far-red light indicated that phytochromes do not regulate the rate or extent of pericarp softening during ripening. Moreover, treatments with red or red/far-red light did not alter the concentrations of citrate, malate, fructose, glucose, or sucrose in fruit. These results are consistent with two conclusions: (a) fruit-localized phytochromes regulate light-induced lycopene accumulation independently of ethylene biosynthesis; and (b) fruit-localized phytochromes are not global regulators of ripening, but instead regulate one or more specific components of this developmental process.     

Arachidonic Acid Alters Tomato HMG Expression and Fruit Growth and Induces 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase-Independent Lycopene Accumulation

Regulation of isoprenoid end-product synthesis required for normal growth and development in plants is not well understood. To investigate the extent to which specific genes for the enzyme 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) are involved in end-product regulation, we manipulated expression of the HMG1 and HMG2 genes in tomato (Lycopersicon esculentum) fruit using arachidonic acid (AA). In developing young fruit AA blocked fruit growth, inhibited HMG1, and activated HMG2 expression. These results are consistent with other reports indicating that HMG1 expression is closely correlated with growth processes requiring phytosterol production. In mature-green fruit AA strongly induced the expression of HMG2, PSY1 (the gene for phytoene synthase), and lycopene accumulation before the normal onset of carotenoid synthesis and ripening. The induction of lycopene synthesis was not blocked by inhibition of HMGR activity using mevinolin, suggesting that cytoplasmic HMGR is not required for carotenoid synthesis. Our results are consistent with the function of an alternative plastid isoprenoid pathway (the Rohmer pathway) that appears to direct the production of carotenoids during tomato fruit ripening.     

Non-destructive assessment of flesh firmness and dietary antioxidants of greenhouse-grown tomato (Solanum lycopersicum L.) at different fruit maturity stages

Non-destructive methods have been widely recognized for evaluating fruit quality traits of many horticultural crops and food processing industry. Destructive (analytical) test, and non-destructive evaluation of the quality traits were investigated and compared for ‘Red Rose’ tomato (Solanum lycopersicum L.) fruit grown under protected environment. Fresh tomato fruit at five distinctive maturity stages namely; breaker (BK), turning (TG), pink (PK), light-red (LR), and red (RD) were labeled and scanned using the handheld near infra-red (NIR) enhanced spectrometer at a wavelength range of 285–1200 nm. The labeled tomato samples were then measured analytically for flesh firmness, lycopene, β-carotene, total phenolic content (TPC) and total flavonoids content (TFC). The results revealed that quality traits could be estimated using NIR spectroscopy with a relatively high coefficient of determination (R²): 0.834 for total phenolic content, 0.864 for lycopene, 0.790 for total flavonoid content, 0.708 for β-carotene; and 0.679 for flesh firmness. The accumulation of Lyco and β-Car rapidly increased in tomatoes harvested between the TG and the LR maturity stages. Harvesting tomatoes at BK maturity stage resulted in significantly higher flesh firmness than harvesting at the later maturity stages. Tomato fruits had the lowest TPC and TFC contents at the earliest maturity stage (BK), while they had intermediate TPC and TFC levels at LR and RD maturity stages. NIR spectroscopic measurements of fruit firmness and lipophilic antioxidants in tomato fruit at various maturity stages were partially in accordance with those estimated by destructive (analytical) methods. Based on these findings, we recommend using non-destructive NIR spectroscopy as an effective tool for predicting tomato fruit quality during harvest stage and postharvest processing.     

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

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 structure and carotenogenic gene expression in red- and white-fleshed loquat (Eriobotrya japonica) fruits

Loquat (Eriobotrya japonica Lindl.) can be sorted into red- and white-fleshed cultivars. The flesh of Luoyangqing (LYQ, red-fleshed) appears red-orange because of a high content of carotenoids while the flesh of Baisha (BS, white-fleshed) appears ivory white due to a lack of carotenoid accumulation. The carotenoid content in the peel and flesh of LYQ was approximately 68 μg g(-1) and 13 μg g(-1) fresh weight (FW), respectively, and for BS 19 μg g(-1) and 0.27 μg g(-1) FW. The mRNA levels of 15 carotenogenesis-related genes were analysed during fruit development and ripening. After the breaker stage (S4), the mRNA levels of phytoene synthase 1 (PSY1) and chromoplast-specific lycopene β-cyclase (CYCB) were higher in the peel, and CYCB and β-carotene hydroxylase (BCH) mRNAs were higher in the flesh of LYQ, compared with BS. Plastid morphogenesis during fruit ripening was also studied. The ultrastructure of plastids in the peel of BS changed less than in LYQ during fruit development. Two different chromoplast shapes were observed in the cells of LYQ peel and flesh at the fully ripe stage. Carotenoids were incorporated in the globules in chromoplasts of LYQ and BS peel but were in a crystalline form in the chromoplasts of LYQ flesh. However, no chromoplast structure was found in the cells of fully ripe BS fruit flesh. The mRNA level of plastid lipid-associated protein (PAP) in the peel and flesh of LYQ was over five times higher than in BS peel and flesh. In conclusion, the lower carotenoid content in BS fruit was associated with the lower mRNA levels of PSY1, CYCB, and BCH; however, the failure to develop normal chromoplasts in BS flesh is the most convincing explanation for the lack of carotenoid accumulation. The expression of PAP was well correlated with chromoplast numbers and carotenoid accumulation, suggesting its possible role in chromoplast biogenesis or interconversion of loquat fruit.     

Identification of novel quantitative trait loci for increased lycopene content and other fruit quality traits in a tomato recombinant inbred line population

Epidemiological and clinical studies indicate that a steady dietary intake of bioavailable lycopene, a C₄₀ carotenoid and potent natural antioxidant, may be associated with a decreased incidence of prostate cancer in humans. Since fresh tomatoes and processed tomato products represent approximately 85% of the average human??s dietary lycopene intake, the identification of novel genetic factors which regulate high fruit lycopene content in tomato is imperative for the improvement of nutritional quality in this commercially valuable specialty crop. To understand the genetic control of the extraordinarily high fruit lycopene content in an accession (LA2093) of the tomato wild species Solanum pimpinellifolium, a quantitative trait locus (QTL) mapping study was conducted using a recombinant inbred line (RIL) population of a cross between LA2093 and a cultivated tomato (S. lycopersicum) breeding line, NCEBR-1. The parental lines, F1 progeny, and F7-F10 RIL populations were grown in replicated field trials in four successive years and evaluated for lycopene content as well as several other traits, including fruit fresh weight, soluble solids content, pH of puree, and plant maturity. The lycopene content of ripe fruit was estimated using three methods: high-performance liquid chromatography (HPLC), spectrophotometry, and colorimetric assays. Based on these measurements, QTL were identified and compared across generations. Among the QTL identified for lycopene, two QTL, located on chromosomes 7 and 12, had very large effects and were consistent across generations. The genomic intervals in which these two QTL reside do not correspond to known map positions of carotenoid biosynthetic genes, indicating that these QTL may represent novel alleles with potentially important implications for tomato breeding as well as increased understanding of carotenoid accumulation in tomato. Several QTL were also identified for fruit weight, soluble solids content and plant maturity. The potential implications of these results for tomato crop improvement are discussed.     

Effect of prolonged root hypoxia on the antioxidant content of tomato fruit

Tomato fruit quality depends on its metabolite content, which in turn is determined by numerous metabolic changes occurring during fruit development and ripening. The aim of this work was to investigate whether flooding affects the nutritional quality of tomato fruit, focusing on compounds essential to human health: carotenoids and ascorbate. To this end, tomato plants (Solanum lycopersicum L. cv Micro-Tom) were submitted to prolonged root hypoxia (1–2% O₂) at first flower anthesis. Fruits were harvested at five stages of the ripening process and analysed for their carotenoid and ascorbate contents. Our results showed that the ripening of fruits that developed on hypoxia treated plants was not inhibited. However, root hypoxia significantly limits carotenoid and ascorbate accumulation in pericarp during fruit ripening, the strongest effects being observed at late stages of ripening. Limitation of both carotenoids and ascorbate accumulation seems to be primarily mediated by the reduced level of expression of genes of the corresponding metabolic pathway.