Ramón Asis

Natural occurring epialleles determine vitamin E accumulation in tomato fruits

Vitamin E (VTE) content is a low heritability nutritional trait for which the genetic determinants are poorly understood. Here, we focus on a previously detected major tomato VTE quantitative trait loci (QTL; mQTL(9-2-6)) and identify the causal gene as one encoding a 2-methyl-6-phytylquinol methyltransferase (namely VTE3(1)) that catalyses one of the final steps in the biosynthesis of γ- and α-tocopherols, which are the main forms of VTE. By reverse genetic approaches, expression analyses, siRNA profiling and DNA methylation assays, we demonstrate that mQTL(9-2-6) is an expression QTL associated with differential methylation of a SINE retrotransposon located in the promoter region of VTE3(1). Promoter DNA methylation can be spontaneously reverted leading to different epialleles affecting VTE3(1) expression and VTE content in fruits. These findings indicate therefore that naturally occurring epialleles are responsible for regulation of a nutritionally important metabolic QTL and provide direct evidence of a role for epigenetics in the determination of agronomic traits.

A candidate gene survey of quantitative trait loci affecting chemical composition in tomato fruit

In tomato, numerous wild-related species have been demonstrated to be untapped sources of valuable genetic variability, including pathogen-resistance genes, nutritional, and industrial quality traits. From a collection of S. pennellii introgressed lines, 889 fruit metabolic loci (QML) and 326 yield-associated loci (YAL), distributed across the tomato genome, had been identified previously. By using a combination of molecular marker sequence analysis, PCR amplification and sequencing, analysis of allelic variation, and evaluation of co-response between gene expression and metabolite composition traits, the present report, provides a comprehensive list of candidate genes co-localizing with a subset of 106 QML and 20 YAL associated either with important agronomic or nutritional characteristics. This combined strategy allowed the identification and analysis of 127 candidate genes located in 16 regions of the tomato genome. Eighty-five genes were cloned and partially sequenced, totalling 45 816 and 45 787 bases from S. lycopersicum and S. pennellii, respectively. Allelic variation at the amino acid level was confirmed for 37 of these candidates. Furthermore, out of the 127 gene-metabolite co-locations, some 56 were recovered following correlation of parallel transcript and metabolite profiling. Results obtained here represent the initial steps in the integration of genetic, genomic, and expressional patterns of genes co-localizing with chemical compositional traits of the tomato fruit.

Genomic Analysis of Wild Tomato Introgressions Determining Metabolism- and Yield-Associated Traits

With the aim of determining the genetic basis of metabolic regulation in tomato fruit, we constructed a detailed physical map of genomic regions spanning previously described metabolic quantitative trait loci of a Solanum pennellii introgression line population. Two genomic libraries from S. pennellii were screened with 104 colocated markers from five selected genomic regions, and a total of 614 bacterial artificial chromosome (BAC)/cosmids were identified as seed clones. Integration of sequence data with the genetic and physical maps of Solanum lycopersicum facilitated the anchoring of 374 of these BAC/cosmid clones. The analysis of this information resulted in a genome-wide map of a nondomesticated plant species and covers 10% of the physical distance of the selected regions corresponding to approximately 1% of the wild tomato genome. Comparative analyses revealed that S. pennellii and domesticated tomato genomes can be considered as largely colinear. A total of 1,238,705 bp from both BAC/cosmid ends and nine large insert clones were sequenced, annotated, and functionally categorized. The sequence data allowed the evaluation of the level of polymorphism between the wild and cultivated tomato species. An exhaustive microsynteny analysis allowed us to estimate the divergence date of S. pennellii and S. lycopersicum at 2.7 million years ago. The combined results serve as a reference for comparative studies both at the macrosyntenic and microsyntenic levels. They also provide a valuable tool for fine-mapping of quantitative trait loci in tomato. Furthermore, they will contribute to a deeper understanding of the regulatory factors underpinning metabolism and hence defining crop chemical composition.

Genetic dissection of vitamin E biosynthesis in tomato

Vegetables are critical for human health as they are a source of multiple vitamins including vitamin E (VTE). In plants, the synthesis of VTE compounds, tocopherol and tocotrienol, derives from precursors of the shikimate and methylerythritol phosphate pathways. Quantitative trait loci (QTL) for α-tocopherol content in ripe fruit have previously been determined in an Solanum pennellii tomato introgression line population. In this work, variations of tocopherol isoforms (α, β, γ, and δ) in ripe fruits of these lines were studied. In parallel all tomato genes structurally associated with VTE biosynthesis were identified and mapped. Previously identified VTE QTL on chromosomes 6 and 9 were confirmed whilst novel ones were identified on chromosomes 7 and 8. Integrated analysis at the metabolic, genetic and genomic levels allowed us to propose 16 candidate loci putatively affecting tocopherol content in tomato. A comparative analysis revealed polymorphisms at nucleotide and amino acid levels between Solanum lycopersicum and S. pennellii candidate alleles. Moreover, evolutionary analyses showed the presence of codons evolving under both neutral and positive selection, which may explain the phenotypic differences between species. These data represent an important step in understanding the genetic determinants of VTE natural variation in tomato fruit and as such in the ability to improve the content of this important nutriceutical.

Transcriptional regulation of tocopherol biosynthesis in tomato.

Tocopherols, compounds with vitamin E (VTE) activity, are potent lipid-soluble antioxidants synthesized only by photosynthetic organisms. Their biosynthesis requires the condensation of phytyl-diphosphate and homogentisate, derived from the methylerythritol phosphate (MEP) and shikimate pathways (SK), respectively. These metabolic pathways are central in plant chloroplast metabolism and are involved in the biosynthesis of important molecules such as chlorophyll, carotenoids, aromatic amino-acids and prenylquinones. In the last decade, few studies have provided insights into the regulation of VTE biosynthesis and its accumulation. However, the pathway regulatory mechanism/s at mRNA level remains unclear. We have recently identified a collection of tomato genes involved in tocopherol biosynthesis. In this work, by a dedicated qPCR array platform, the transcript levels of 47 genes, including paralogs, were determined in leaves and across fruit development. Expression data were analyzed for correlation with tocopherol profiles by coregulation network and neural clustering approaches. The results showed that tocopherol biosynthesis is controlled both temporally and spatially however total tocopherol content remains constant. These analyses exposed 18 key genes from MEP, SK, phytol recycling and VTE-core pathways highly associated with VTE content in leaves and fruits. Moreover, genomic analyses of promoter regions suggested that the expression of the tocopherol-core pathway genes is trancriptionally coregulated with specific genes of the upstream pathways. Whilst the transcriptional profiles of the precursor pathway genes would suggest an increase in VTE content across fruit development, the data indicate that in the M82 cultivar phytyl diphosphate supply limits tocopherol biosynthesis in later fruit stages. This is in part due to the decreasing transcript levels of geranylgeranyl reductase (GGDR) which restricts the isoprenoid precursor availability. As a proof of concept, by analyzing a collection of Andean landrace tomato genotypes, the role of the pinpointed genes in determining fruit tocopherol content was confirmed. The results uncovered a finely tuned regulation able to shift the precursor pathways controlling substrate influx for VTE biosynthesis and overcoming endogenous competition for intermediates. The whole set of data allowed to propose that 1-deoxy-D-xylulose-5-phosphate synthase and GGDR encoding genes, which determine phytyl-diphosphate availability, together with enzyme encoding genes involved in chlorophyll-derived phytol metabolism appear as the most plausible targets to be engineered aiming to improve tomato fruit nutritional value.

Determination of Aflatoxin B 1 in Highly Contaminated Peanut Samples Using HPLC and ELISA

The precision, accuracy, detection limit and peanut matrix influence of an ELISA were analysed in the determination of aflatoxin B 1 . The assay was performed on two different reference samples: peanut extract and peanut paste, that were spiked with known amounts of aflatoxins. The lower detectable level was 0.5 w g kg -1 . The average intra-assay precision expressed as coefficient of variation (CV) was 11.7% for concentrations between 2.54 and 901 w g kg -1 and the average inter-assay precision was 29.7% for the same range of concentrations. The average accuracy measured by a recovery assay in samples that contained only aflatoxin B 1 was 107%. The correlation between the ELISA and high-performance liquid chromatography (HPLC) applied to 28 peanut samples artificially contaminated with Aspergillus flavus and A. parasiticus spores showed a high correlation ( r = 0.977, P < 0.0001). The detection limits and matrix influence associated with our ELISA procedure were more sensitive than those reported for other ELISA procedures due to specific antiserum treatment.

Aflatoxin production in six peanut (Arachis hypogaea L.) genotypes infected with Aspergillus flavus and Aspergillus parasiticus, isolated from peanut production areas of Cordoba, Argentina.

Aflatoxin contamination is one of the main factors affecting peanut seed quality. One of the strategies to decrease the risk of peanut aflatoxin contamination is the use of genotypes with resistance to Aspergillus infection. This laboratory study reports the resistance to Aspergillus infection and aflatoxin contamination of six peanut genotypes inoculated with 21 Aspergillus isolates obtained from the peanut production region of Cordoba, Argentina. The resistance was investigated in the seed coat and cotyledons of three resistant genotypes (J11, PI 337394, and PI 337409) and three breeding lines (Manfredi 68, Colorado Irradiado, and Florman INTA) developed at the Instituto Nacional de Tecnologia Agropecuaria (INTA), Manfredi Experimental Station, Cordoba, Argentina. Resistance to fungal colonization and aflatoxin contamination was found to be associated with seed coat integrity in the PI 337394, PI 337409, and J11 genotypes, whereas the INTA breeding lines such as Colorado Irradiado showed a moderate resistance and the Manfredi 68 and Florman INTA genotypes the least resistance. Furthermore, another type of resistance associated with cotyledons was found only in the PI 337394 genotype.

Oxidative stress response induced by atrazine in Palaemonetes argentinus: the protective effect of vitamin E.

The widespread contamination and persistence of the herbicide atrazine residues in the environment resulted in the exposure of non-target organisms. The present study was undertaken to investigate the effect of atrazine in the response of oxidative stress biomarkers in the freshwater shrimp Palaemonetes argentinus and the protective effect of vitamin-E against atrazine-induced toxicity. Therefore, two batches of P. argentinus were fed for 21 days with a commercial food enriched in proteins (D1) or with D2, composed of D1 enriched with vitamin-E (6.8 and 16.0mg% of vitamin-E, respectively). Subsequently, half of the individuals of each group were exposed to atrazine (0.4mgL(-1)) for 24h and the others remained as controls. Atrazine promoted oxidative stress response in P. argentinus fed with D1 as indicated by enhanced H2O2 content and induction of superoxide dismutase, glutathione-S-transferases and glutathione reductase. This antioxidant activity would prevent the increment of thiobarbituric acid reactive substances in the shrimp tissues. P. argentinus fed with D2 reversed the response of the biomarkers measured. However, the activation of antioxidants response had an energetic cost, which was revealed by a decrease in lipids storage in shrimps. These results show the modulatory effect of vit-E on oxidative stress and its potential use as an effective antioxidant to be applied in chemoprotection strategies during aquaculture.

Hydrophilic antioxidants from Andean tomato landraces assessed by their bioactivities in vitro and in vivo

Potential nutraceutical properties of hydrophilic antioxidants in fruits of tomato landraces collected in Andean valleys were characterised. Antioxidant metabolites were measured by HPLC-DAD-MS/MS in mature fruits and their biological activities were assessed by in vitro and in vivo methods. In vitro antioxidant capacities were established by TEAC and FRAP methods. For in vivo biological activities we used a procedure based on Caenorhabditis elegans subjected to thermal stress. In addition, Saccharomyces cerevisiae was also used as a rapid screening system to evaluate tomato antioxidant capacity. All tomato accessions displayed significant differences regarding metabolic composition, biological activity and antioxidant capacity. Metabolite composition was associated with geographical origin and fruit size. Antioxidant activities showed significant association with phenolic compounds, such as caffeoylquinic acids, ferulic acid-O-hexosides and rutin. Combination of in vitro and in vivo methods applied here allowed evaluation of the variability in nutraceutical properties of tomato landraces, which could be applied to other fruits or food products.

Down-regulation of tomato PHYTOL KINASE strongly impairs tocopherol biosynthesis and affects prenyllipid metabolism in an organ-specific manner

Highlight Phytol kinase plays a key role in the regulation of isoprenoid metabolism in an organ-specific manner.