Ana-Rosa Ballester

Biochemical and Molecular Analysis of Pink Tomatoes: Deregulated Expression of the Gene Encoding Transcription Factor SlMYB12 Leads to Pink Tomato Fruit Color

The color of tomato fruit is mainly determined by carotenoids and flavonoids. Phenotypic analysis of an introgression line (IL) population derived from a cross between Solanum lycopersicum ‘Moneyberg’ and the wild species Solanum chmielewskii revealed three ILs with a pink fruit color. These lines had a homozygous S. chmielewskii introgression on the short arm of chromosome 1, consistent with the position of the y (yellow) mutation known to result in colorless epidermis, and hence pink-colored fruit, when combined with a red flesh. Metabolic analysis showed that pink fruit lack the ripening-dependent accumulation of the yellow-colored flavonoid naringenin chalcone in the fruit peel, while carotenoid levels are not affected. The expression of all genes encoding biosynthetic enzymes involved in the production of the flavonol rutin from naringenin chalcone was down-regulated in pink fruit, suggesting that the candidate gene underlying the pink phenotype encodes a regulatory protein such as a transcription factor rather than a biosynthetic enzyme. Of 26 MYB and basic helix-loop-helix transcription factors putatively involved in regulating transcription of genes in the phenylpropanoid and/or flavonoid pathway, only the expression level of the MYB12 gene correlated well with the decrease in the expression of structural flavonoid genes in peel samples of pink- and red-fruited genotypes during ripening. Genetic mapping and segregation analysis showed that MYB12 is located on chromosome 1 and segregates perfectly with the characteristic pink fruit color. Virus-induced gene silencing of SlMYB12 resulted in a decrease in the accumulation of naringenin chalcone, a phenotype consistent with the pink-colored tomato fruit of IL1b. In conclusion, biochemical and molecular data, gene mapping, segregation analysis, and virus-induced gene silencing experiments demonstrate that the MYB12 transcription factor plays an important role in regulating the flavonoid pathway in tomato fruit and suggest strongly that SlMYB12 is a likely candidate for the y mutation.

Metabolite biodiversity in pepper (Capsicum) fruits of thirty-two diverse accessions: Variation in health-related compounds and implications for breeding

A comprehensive study on morphology and biochemical compounds of 32 Capsicum spp. accessions has been performed. Accessions represented four pepper species, Capsicum annuum, Capsicum frutescens, Capsicum chinense and Capsicum baccatum which were selected by their variation in morphological characters such as fruit color, pungency and origin. Major metabolites in fruits of pepper, carotenoids, capsaicinoids (pungency), flavonoid glycosides, and vitamins C and E were analyzed and quantified by high performance liquid chromatography. The results showed that composition and level of metabolites in fruits varied greatly between accessions and was independent of species and geographical location. Fruit color was determined by the accumulation of specific carotenoids leading to salmon, yellow, orange, red and brown colored fruits. Levels of both O- and C-glycosides of quercetin, luteolin and apigenin varied strongly between accessions. All non-pungent accessions were devoid of capsaicins, whereas capsaicinoid levels ranged from 0.07 up to 80 mg/100g fr. wt. in fruit pericarp. In general, pungent accessions accumulated the highest capsaicinoid levels in placenta plus seed tissue compared to pericarp. The non-pungent capsaicinoid analogs, capsiates, could be detected at low levels in some pungent accessions. All accessions accumulated high levels of vitamin C, up to 200 mg/100g fr. wt. The highest vitamin E concentration found was 16 mg/100g fr. wt. Based on these metabolic data, five accessions were selected for further metabolic and molecular analysis, in order to isolate key genes involved in the production of these compounds and to assist future breeding programs aimed at optimizing the levels of health-related compounds in pepper fruit.

Genome sequence of the necrotrophic fungus Penicillium digitatum, the main postharvest pathogen of citrus

BackgroundPenicillium digitatum is a fungal necrotroph causing a common citrus postharvest disease known as green mold. In order to gain insight into the genetic bases of its virulence mechanisms and its high degree of host-specificity, the genomes of two P. digitatum strains that differ in their antifungal resistance traits have been sequenced and compared with those of 28 other Pezizomycotina.ResultsThe two sequenced genomes are highly similar, but important differences between them include the presence of a unique gene cluster in the resistant strain, and mutations previously shown to confer fungicide resistance. The two strains, which were isolated in Spain, and another isolated in China have identical mitochondrial genome sequences suggesting a recent worldwide expansion of the species. Comparison with the closely-related but non-phytopathogenic P. chrysogenum reveals a much smaller gene content in P. digitatum, consistent with a more specialized lifestyle. We show that large regions of the P. chrysogenum genome, including entire supercontigs, are absent from P. digitatum, and that this is the result of large gene family expansions rather than acquisition through horizontal gene transfer. Our analysis of the P. digitatum genome is indicative of heterothallic sexual reproduction and reveals the molecular basis for the inability of this species to assimilate nitrate or produce the metabolites patulin and penicillin. Finally, we identify the predicted secretome, which provides a first approximation to the protein repertoire used during invasive growth.ConclusionsThe complete genome of P. digitatum, the first of a phytopathogenic Penicillium species, is a valuable tool for understanding the virulence mechanisms and host-specificity of this economically important pest.

Genome, Transcriptome, and Functional Analyses of Penicillium expansum Provide New Insights Into Secondary Metabolism and Pathogenicity

The relationship between secondary metabolism and infection in pathogenic fungi has remained largely elusive. The genus Penicillium comprises a group of plant pathogens with varying host specificities and with the ability to produce a wide array of secondary metabolites. The genomes of three Penicillium expansum strains, the main postharvest pathogen of pome fruit, and one Pencillium italicum strain, a postharvest pathogen of citrus fruit, were sequenced and compared with 24 other fungal species. A genomic analysis of gene clusters responsible for the production of secondary metabolites was performed. Putative virulence factors in P. expansum were identified by means of a transcriptomic analysis of apple fruits during the course of infection. Despite a major genome contraction, P. expansum is the Penicillium species with the largest potential for the production of secondary metabolites. Results using knockout mutants clearly demonstrated that neither patulin nor citrinin are required by P. expansum to successfully infect apples. Li et al. ( MPMI-12-14-0398-FI ) reported similar results and conclusions in their recently accepted paper.

Differential Tomato Transcriptomic Responses Induced by Pepino Mosaic Virus Isolates with Differential Aggressiveness

Pepino mosaic virus (PepMV) is a highly infectious potexvirus and a major disease of greenhouse tomato (Solanum lycopersicum) crops worldwide. Damage and economic losses caused by PepMV vary greatly and can be attributed to differential symptomatology caused by different PepMV isolates. Here, we used a custom-designed Affymetrix tomato GeneChip array with probe sets to interrogate over 22,000 tomato transcripts to study transcriptional changes in response to inoculation of tomato seedlings with a mild and an aggressive PepMV isolate that share 99.4% nucleotide sequence identity. The two isolates induced a different transcriptomic response, despite accumulating to similar viral titers. PepMV inoculation resulted in repression of photosynthesis. In addition, defense responses were stronger upon inoculation with the aggressive isolate, in both cases mediated by salicylic acid signaling rather than by jasmonate signaling. Our results furthermore show that PepMV differentially regulates the RNA silencing pathway, suggesting a role for a PepMV-encoded silencing suppressor. Finally, perturbation of pigment biosynthesis, as shown by differential regulation of the flavonoid and lycopene biosynthesis pathways, was monitored. Metabolite analyses on mature fruits of PepMV-infected tomato plants, which showed typical fruit marbling, revealed a decrease in carotenoids, likely responsible for the marbled phenotype, and an increase in alkaloids and phenylpropanoids that are associated with pathogen defense in the yellow sectors of the fruit.

Metabolomics and molecular marker analysis to explore pepper (Capsicum sp.) biodiversity

An overview of the metabolic diversity in ripe fruits of a collection of 32 diverse pepper (Capsicum sp.) accessions was obtained by measuring the composition of both semi-polar and volatile metabolites in fruit pericarp, using untargeted LC–MS and headspace GC–MS platforms, respectively. Accessions represented C. annuum, C. chinense, C. frutescens and C. baccatum species, which were selected based on variation in morphological characters, pungency and geographic origin. Genotypic analysis using AFLP markers confirmed the phylogenetic clustering of accessions according to Capsicum species and separated C. baccatum from the C. annuum–C. chinense–C. frutescens complex. Species-specific clustering was also observed when accessions were grouped based on their semi-polar metabolite profiles. In total 88 semi-polar metabolites could be putatively identified. A large proportion of these metabolites represented conjugates of the main pepper flavonoids (quercetin, apigenin and luteolin) decorated with different sugar groups at different positions along the aglycone. In addition, a large group of acyclic diterpenoid glycosides, called capsianosides, was found to be highly abundant in all C. annuum genotypes. In contrast to the variation in semi-polar metabolites, the variation in volatiles corresponded well to the differences in pungency between the accessions. This was particularly true for branched fatty acid esters present in pungent accessions, which may reflect the activity through the acyl branch of the metabolic pathway leading to capsaicinoids. In addition, large genetic variation was observed for many well-established pepper aroma compounds. These profiling data can be used in breeding programs aimed at improving metabolite-based quality traits such as flavour and health-related metabolites in pepper fruits.

Secondary Metabolites of Capsicum Species and Their Importance in the Human Diet

The genus Capsicum (pepper) comprises a large number of wild and cultivated species. The plants are grown all over the world, primarily in tropical and subtropical countries. The fruits are an excellent source of health-related compounds, such as ascorbic acid (vitamin C), carotenoids (provitamin A), tocopherols (vitamin E), flavonoids, and capsaicinoids. Pepper fruits have been used for fresh and cooked consumption, as well as for medicinal purposes, such as treatment of asthma, coughs, sore throats, and toothache. Depending on its uses, there are several main characters important for product quality; pungency, bright attractive colors, highly concentrated extracts, and a small number of seeds are the main characters on which quality is based and priced. Herein, a general overview of biochemical composition, medical properties of these compounds, and characteristics of quality attributes of pepper fruits is presented.

Transcriptomic profiling of citrus fruit peel tissues reveals fundamental effects of phenylpropanoids and ethylene on induced resistance

Penicillium spp. are the major postharvest pathogens of citrus fruit in Mediterranean climatic regions. The induction of natural resistance constitutes one of the most promising alternatives to avoid the environmental contamination and health problems caused by chemical fungicides. To understand the bases of the induction of resistance in citrus fruit against Penicillium digitatum, we have used a 12k citrus cDNA microarray to study transcriptional changes in the outer and inner parts of the peel (flavedo and albedo, respectively) of elicited fruits. The elicitor treatment led to an over-representation of biological processes associated with secondary metabolism, mainly phenylpropanoids and cellular amino acid biosynthesis and methionine metabolism, and the down-regulation of genes related to biotic and abiotic stresses. Among phenylpropanoids, we detected the over-expression of a large subset of genes important for the synthesis of flavonoids, coumarins and lignin, especially in the internal tissue. Furthermore, these genes and those of ethylene biosynthesis showed the highest induction. The involvement of both phenylpropanoid and ethylene pathways was confirmed by examining changes in gene expression and ethylene production in elicited citrus fruit. Therefore, global results indicate that secondary metabolism, mainly phenylpropanoids, and ethylene play important roles in the induction of resistance in citrus fruit.

Citrus phenylpropanoids and defence against pathogens. Part I: metabolic profiling in elicited fruits.

Penicillium spp. are among the major postharvest pathogens of citrus fruit. Induction of natural resistance in fruits constitutes one of the alternatives to chemical fungicides. Here, we investigated the involvement of the phenylpropanoid pathway in the induction of resistance in Navelate oranges by examining changes in the metabolic profile of upon eliciting citrus fruits. By using both HPLC-PDA-FD and HPLC-PDA-QTOF-MS allowed the identification of several compounds that seem to be relevant for induced resistance. In elicited fruits, a greater diversity of phenolic compounds was observed in the flavedo (outer coloured part of the peel) when compared to the albedo (inner white part). Moreover, only small changes were detected in the most abundant citrus flavonoids. The coumarin scoparone was among the compounds with the highest induction upon elicitation. Two other highly induced compounds were identified as citrusnin A and drupanin aldehyde. All three compounds are known to exert antimicrobial activity. Our results suggest that phenylpropanoids and their derivatives play an important role in the induction of resistance in citrus fruit.

Citrus phenylpropanoids and defence against pathogens. Part II: gene expression and metabolite accumulation in the response of fruits to Penicillium digitatum infection.

The effect of infection of Citrus sinensis (var. Navelina) fruits with Penicillium digitatum was studied at gene expression and metabolite levels. In this study, expression of genes involved in the phenylpropanoid pathway was studied in the flavedo (outer coloured part of the peel) and albedo (inner white part) in response to pathogen infection. Results of the time-course experiment showed that maximal expression of 10 out of 17 phenylpropanoid genes analysed occurred at 48h post-inoculation, when decay symptoms started to appear, and mRNA levels either kept constant or decreased after 72h post-inoculation. To further investigate the putative involvement of the phenylpropanoid pathway in the defence of citrus fruit, changes in the metabolic profile of both tissues infected with P. digitatum was studied by means of HPLC-PDA-FD. Metabolite accumulation levels along the time course suggest that flavanones, flavones, polymethoxylated flavones and scoparone are induced in citrus fruit in response to P. digitatum infection, although with different trends depending on the tissue.