Pectic enzymes as potential enhancers of ascorbic acid production through the D-galacturonate pathway in Solanaceae

The increase of L-Ascorbic Acid (AsA) content in tomato (Solanum lycopersicum) is a common goal in breeding programs due to its beneficial effect on human health. To shed light into the regulation of fruit AsA content, we exploited a Solanum pennellii introgression line (IL12-4-SL) harbouring one quantitative trait locus that increases the content of total AsA in the fruit. Biochemical and transcriptomic analyses were carried out in fruits of IL12-4-SL in comparison with the cultivated line M82 at different stages of ripening. AsA content was studied in relation with pectin methylesterase (PME) activity and the degree of pectin methylesterification (DME). Our results indicated that the increase of AsA content in IL12-4-SL fruits was related with pectin de-methylesterification/degradation. Specific PME, polygalacturonase (PG) and UDP-D-glucuronic-acid-4-epimerase (UGlcAE) isoforms were proposed as components of the D-galacturonate pathway leading to AsA biosynthesis. The relationship between AsA content and PME activity was also exploited in PMEI tobacco plants expressing a specific PME inhibitor (PMEI). Here we report that tobacco PMEI plants, altered in PME activity and degree of pectin methylesterification, showed a reduction in low methylesterified pectic domains and exhibited a reduced AsA content. Overall, our results provide novel biochemical and genetic traits for increasing antioxidant content by marker-assisted selection in the Solanaceae family

Positive selection in the leucine-rich repeat domain of Gro1 genes in Solanum species

In pathogen resistant plants, solvent-exposed residues in the leucine-rich repeat (LRR) proteins are thought to mediate resis- tance by recognizing plant pathogen elicitors. In potato, the gene Gro1-4 confers resistance to Globodera rostochiensis. The investigation of variablity in different copies of this gene represents a good model for the verification of positive selection mechanisms. Two datasets of Gro1 LRR sequences were constructed, one derived from the Gro1-4 gene, belonging to dif- ferent cultivated and wild Solanum species, and the other belonging to paralogues of a resistant genotype. Analysis of non- synonymous to synonymous substitution rates (Ka/Ks) highlighted 14 and six amino acids with Ka/Ks >1 in orthologue and paralogue datasets, respectively. Selection analysis revealed that the leucine-rich regions accumulate variability in a very specific way, and we found that some combinations of amino acids in these sites might be involved in pathogen recognition. The results confirm previous studies on positive selection in the LRR domain of R protein in Arabidopsis and other model plants and extend these to wild Solanum species. Moreover, positively selected sites in the Gro1 LRR domain show that coevolution mainly occurred in two regions on the internal surface of the three-dimensional horseshoe structure of the domain, albeit with different evolutionary forces between paralogues and orthologues

‘Omics’ approaches in tomato aimed at identifying candidate genes for ascorbic acid accumulation in the fruit

Tomato (Solanum lycopersicum) is one of the most important vegetables in the world with significant importance for human health and nutrition. This species has long served as model system for plant genetics, development, physiology, pathology, and fleshy fruit ripening, resulting in the accumulation of many genetic and genomic resources. In addition, the tremendous development of high-throughput technologies, such as transcriptomics, metabolomics and proteomics, collectively denoted as ‘omics’ technologies, has led to a huge collection of data and platforms today available on the net. Nowadays, identifying all the components of a single biological system is within our means; however, assigning function to genes, proteins and metabolites is still a daunting task. Major challenges include interpretation and integration of large datasets to understand the principles underlying the regulation of genes, metabolites and proteins, and how their combined interactions associate with variation in phenotype. In this review, we will focus on the role of the different high-throughput technologies in enhancing tomato breeding particularly for fruit quality traits. We also describe how two ‘omics’ approaches could be combined in order to identify candidate genes for the genetic control of ascorbic acid accumulation in tomato fruit. We report the example of transcriptomic and genomic approaches established on the use of different high-throughput platforms available for tomato.Key words: Tomato, introgression lines, quality trait, genomics, transcriptomics, candidate gene, single nucleotide polymorphism (SNPs)

Resistance to Ralstonia Solanacearum of sexual hybrids between Solanum commersonii and S. tuberosum

This research was carried out to study the levels of bacterial wilt resistance and genetic diversity of (near) pentaploid sexual hybrids between S. commersonii (2n = 2x = 24, 1EBN) and cultivated S. tuberosum. Following artificial inoculations with Ralstonia solanacearum, wilting degree was estimated on a scale from 0 to 4, and seven genotypes of 26 (27%) displaying a S. commersonii like behavior were identified. Latent bacterial colonizations were detected in roots of symptomless S. commersonii and hybrids, whereas no bacterial populations were detected within stems. This suggests that the movement and/or growth of the bacterium in the aerial part were strongly inhibited. A molecular study with AFLP markers clustered hybrids into nine groups and provided evidence that resistant hybrids were slightly more similar to cultivated S. tuberosum than to the wild parent. This is important in view of the re-establishment of the cultivated genetic background through backcrosses. Hybrids displayed good fertility and are being used for further breeding efforts

Enhancing the Health-Promoting Effects of Tomato Fruit for Biofortified Food

Consumption of tomato fruits, like those of many other plant species that are part of the human diet, is considered to be associated with several positive effects on health. Indeed, tomato fruits are an important source of bioactive compounds with known beneficial effects including vitamins, antioxidants, and anticancer substances. In particular, antioxidant metabolites are a group of vitamins, carotenoids, phenolic compounds, and phenolic acid that can provide effective protection by neutralizing free radicals, which are unstable molecules linked to the development of a number of degenerative diseases and conditions. In this review, we will summarize the recent progress on tomatoes nutritional importance and mechanisms of action of different phytochemicals against inflammation processes and prevention of chronic noncommunicable diseases (e.g., obesity, diabetes, coronary heart disease, and hypertension). In addition, we will summarize the significant progress recently made to improve the nutritional quality of tomato fruits through metabolic engineering and/or breeding

New insights in the control of antioxidants accumulation in tomato by transcriptomic analyses of genotypes exhibiting contrasting levels of fruit metabolites

Abstract Background Tomato is an economically important crop with fruits that are a significant source of bioactive compounds such as ascorbic acid and phenolics. Nowadays, the majority of the enzymes of the biosynthetic pathways and of the structural genes controlling the production and the accumulation of antioxidants in plants are known; however, the mechanisms that regulate the expression of these genes are yet to be investigated. Here, we analyzed the transcriptomic changes occurring during ripening in the fruits of two tomato cultivars (E1 and E115), characterized by a different accumulation of antioxidants, in order to identify candidate genes potentially involved in the biosynthesis of ascorbic acid and phenylpropanoids. Results RNA sequencing analyses allowed identifying several structural and regulator genes putatively involved in ascorbate and phenylpropanoids biosynthesis in tomato fruits. Furthermore, transcription factors that may control antioxidants biosynthesis were identified through a weighted gene co-expression network analysis (WGCNA). Results obtained by RNA-seq and WGCNA analyses were further confirmed by RT-qPCR carried out at different ripening stages on ten cultivated tomato genotypes that accumulate different amount of bioactive compounds in the fruit. These analyses allowed us to identify one pectin methylesterase, which may affect the release of pectin-derived D-Galacturonic acid as metabolic precursor of ascorbate biosynthesis. Results reported in the present work allowed also identifying one L-ascorbate oxidase, which may favor the accumulation of reduced ascorbate in tomato fruits. Finally, the pivotal role of the enzymes chalcone synthases (CHS) in controlling the accumulation of phenolic compounds in cultivated tomato genotypes and the transcriptional control of the CHS genes exerted by Myb12 were confirmed. Conclusions By using transcriptomic analyses, candidate genes encoding transcription factors and structural genes were identified that may be involved in the accumulation of ascorbic acid and phenylpropanoids in tomato fruits of cultivated genotypes. These analyses provided novel insights into the molecular mechanisms controlling antioxidants accumulation in ripening tomato fruits. The structural genes and regulators here identified could also be used as efficient genetic markers for selecting high antioxidants tomato cultivars

Evaluation of Tomato Genetic Resources for Response to Water Deficit

Water deficit strongly affects plant yield and quality. However, plants can minimize drought injury by adaptation mechanisms that have evolved to escape harmful conditions. The response to water deprivation is a complex trait controlled by several genes. In order to gain a deeper understanding of drought response mechanisms in tomato, a collection of 27 genotypes was studied under different water deficit conditions. Since developmental stages might be differently influenced by drought, analyses were carried out on young plantlets during fruit setting. The only genotype that showed good performances both as water retention and fruit production was the ecotype Siccagno. All the genotypes were analysed at molecular level with the aim of detecting structural polymorphisms in selected stress-responsive genes. In addition, the expression level of a number of these genes was measured in the genotypes more tolerant to water deficit. Many polymorphisms were detected in six stress-responsive genes, and some could imply significant modifications in the protein structure. Furthermore, the expression analysis by RT-qPCR of three stress-responsive genes allowed arguing that a higher level of expression of the gene erd15 might be related to the better response to water deficit exhibited by Siccagno. Similarly, the lower expression of eight genes in the same genotype analysed through a microarray experiment confirmed the involvement of these stress-related genes in the tomato response to drought. Further investigations are required for a better comprehension of the mechanisms underlying response to water deficit in tomato by exploiting the genetic resource identified as more tolerant. The use of new technologies able to globally analyse structural polymorphism and expression level of genes will succeed to identify crucial genes involved in stress response in the ecotype Siccagno grown under different water regimes

Comparative transcriptomic profiling of two tomato lines with different ascorbate content in tomato fruit.

In recent years, interest in tomato breeding for enhanced antioxidant content has increased as medical research has pointed to human health benefits from antioxidant dietary intake. Ascorbate is one of the major antioxidants present in tomato, and little is known about mechanisms governing ascorbate pool size in this fruit. In order to provide further insights into genetic mechanisms controlling ascorbate biosynthesis and accumulation in tomato, we investigated the fruit transcriptome profile of the Solanum pennellii introgression line 10-1 that exhibits a lower fruit ascorbate level than its cultivated parental genotype. Our results showed that this reduced ascorbate level is associated with an increased antioxidant demand arising from an accelerated oxidative metabolism mainly involving mitochondria, peroxisomes, and cytoplasm. Candidate genes for controlling ascorbate level in tomato fruit were identified, highlighting the role of glycolysis, glyoxylate metabolism, and purine breakdown in modulating the ascorbate pool size

A basic Helix-Loop-Helix (SlARANCIO), identified from a Solanum pennellii introgression line, affects carotenoid accumulation in tomato fruits

Abstract Carotenoid accumulation in tomato (Solanum lycopersicum) fruits is influenced by environmental stimuli and hormonal signals. However, information on the relative regulatory mechanisms are scanty since many molecular players of the carotenoid biosynthetic pathway are still unknown. Here, we reported a basic Helix-Loop-Helix transcription factor, named SlARANCIO (SlAR), whose silencing influences carotenoid accumulation in tomato fruits. The SlAR gene was found in the S. pennellii introgression line (IL) 12-4SL that holds the carotenoid QTL lyc12.1. We observed that the presence of the wild region in a cultivated genetic background led to a decrease in total carotenoid content of IL12-4SL fruits. To get insights into the function of SlAR, a quick reverse genetic approach was carried out. Virus-induced gene silencing of SlAR in S. lycopersicum M82 and MicroTom fruits reproduced the same phenotype observed in IL12-4SL, i.e. decreased content of lycopene and total carotenoids. Vice versa, the overexpression of SlAR in Nicotiana benthamiana leaves increased the content of total carotenoids and chlorophylls. Our results, combined with public transcriptomic data, highly suggest that SlAR acts indirectly on the carotenoid pathway and advances current knowledge on the molecular regulators controlling lyc12.1 and, potentially, precursors of carotenoid biosynthesis