Tomato ionomic approach for food fortification and safety.

Food fortification is an issue of paramount of importance for people living both in developed and in developing countries. Among substances listed as "nutriceuticals", essential minerals have been recognised for their involvement in several healthy issues, involving all ages. In this frame, food plants are playing a pivotal role since their capability to compartmentalise ions and proteinmetal complexes in edible organs. Conversely, the accumulation of high metal levels in those organs may lead to safety problems. In the recent years, thanks to the availability of new and improved analytical apparatus in both ionic and genomic/transcrittomics areas, it is became feasible to couple data coming from plant physiology and genetics. Ionomics is the discipline that studies the cross-analysis of both data sets. Our group, in the frame of GenoPom project granted by MiUR, is interested to study the ionomics of tomatoes cultivars derived by breeding programmes in which wild relatives have been used to transfer several useful traits, such as resistance to biotic or abiotic stresses, fruit composition and textiture, etc. The introgression of the wild genome into the cultivated one produces new gene combinations. They might lead to the expression of some traits, such as increased or reduced adsorption of some metals and their exclusion or loading into edible organs, thus strongly involving the nutritional food value. Our final goal is to put together data coming from ions homeostasis and gene expression analyses, thus obtaining an ionomic tomato map related to ions absorption, translocation and accumulation in various plant organs, fruits included. To follow our hypothesis, we are studying the ionome of Solanum lycopersicum cv. M82 along with 76 Introgression Lines (ILs) produced by interspecific crosses between this cultivar and the wild species S. pennellii. These ILs are homozygous for small portions of the wild species genome introgressed into the domesticated M82 one. They are used as a useful tool for mapping QTL associated with many traits of interest. It is worthy to note that, until now, little information is available on QTL for ions accumulation in tomato. Moreover, as our knowledge, effects of new gene combinations in introgressed lines on ions uptake related to food safety have not been extensively studied. In this presentation we show results coming from the ionome analysis, carried out on S . lycopersicum M82 and several ILs. Plants were grown in pots in a greenhouse and watered with deionised water Thirty day-old plants were left to grow for 15 days in the presence of non-toxic concentration of Cd, Pb, As, Cr and Zn given combined. Leaves of all plants were then harvested and stored at -80°C for ionome and gene expression analyses. Preliminary results of ionome analysis of S. lycopersicum M82 and several ILs, carried out using an ICP-MS, showed that traits correlated to toxic metals and micronutrients accumulation in apical leaves were significantly modified in response to specific genetic backgrounds. Those results are perhaps due to the introgression of traits linked to uptake, translocation and accumulation of useful and/or toxic metal into plant apical leaves and to interactions of the wild type introgressed genomic regions with the cultivated genome. Also, data are shown on the identification and isolation of Solanum gene sequences related to ions uptake, translocation and accumulation, useful for further real-time gene expression evaluation in both cultivated and ILs during the treatments with the above-mentioned metals

Validation of an Eco-Friendly Automated Method for the Determination of Glucose and Fructose in Wines

Fermentable sugar dosage helps oenologists to establish a harvest’s moment and control the fermentation process of the musts. The official analyses recommended for their determination are long, laborious, and must be carried out by specialized personnel. On the contrary, instrumental analysis automation limits human errors, increases precision, and reduces the time and cost of the analyses. In the food production sector, to use methods other than those recommended by supranational bodies in official reports, it is necessary to validate the analytical processes to establish the conformity of the results between the new methods and the reference ones. This work validated an automated enzymatic apparatus to determine the sum of glucose and fructose levels in wine samples. The validation was carried out on wine samples (dry red wine, dry white wine, moderately sweet wine, and sweet wine) containing different sugar concentrations by comparing data obtained using the OIV-MA-AS311-02 method performed by a specialized operator (reference method) and the same method performed by an automated apparatus. The difference between the results’ means obtained with the two procedures was significant. Nevertheless, the automated procedure was considered suitable for the intended use since the differences between the averages were lower than the measurement uncertainty at the same concentration, and the repeatability results were better for the automated procedure than the reference method

SOLANUM LYCOPERSICUM X S. PENNELLII INTROGRESSION LINES WERE USEFUL TO CHARACTERISE THE IONOME OF TOMATO FRUIT

In the frame of "GenoPom" PON-MIUR project, we have began a study of tomato ionome in order to identify the contribution of specific chromosome and part of them on the ionome. For this purpose, we have analysed by ICP-MS plants of an introgression line (IL) population derived from the cross between Solanum lycopersicum cv. M82 and S. pennellii (Eshed and Zamir, 1995) grown under controlled environmental conditions.we report results derived from tomato whole fruits ionome analysis of 30 IL, covering all 12 tomato chromosomes, along with the recurrent parent cv. M82. Among several detected elements, the following ones have firstly been analysed: Ca, Fe, Cu, Zn and Se. Each element concentration data were referred to cv. M82

Ion uptake and YSL1 gene identification in tomato

Tomato breeder are using wild tomato relatives, even non-cross compatibles ones, in order to obtain cultivars with highly commercial values bearing new traits. However, the introgression of a wild genome into the cultivated one produces a new gene combinations that may lead to the expression of undeliverable traits, perhaps not so easy to recognise; even more, phenotypic variations may escape during the selection procedure when minor genes or non-abnormal phenotypes are involved. In the frame of the “GenoPom” project funded by MIUR, we have focused our interest on the alteration of heavy metals uptake from the soil and their loading into edible organs in commercial lines coming from Solanum interspecific crosses. Our final aim is to put together data coming from ion homeostasis and gene expression analyses, thus obtaining a ionomic map of tomato. To pursue our goal, we have started to study the cv M82 of Solanum lycopersicon, the wild relative Solanum pennelli and their introgression lines IL. Regarding the experiments on ion homeostasis, S. lycopersicon M82 and the introgression line IL 6-4-2 were grown in hydroponics under controlled environmental conditions. Twenty day-old plants were left to grow for 10 days in the presence of non-toxic concentration of Cd (10 mM), Pb (3 mM), Zn (100 mM) given separately or combined. Control and treated roots and leaves were then harvested and stored at -80°C for ionic and gene expression analyses. Ions analysis of Solanum lycopersicon M82 and IL 6-4-2 showed that traits correlated to ionic homeostasis is significantly modified in response to all metals and to the genotype. The analysis of ions data, obtained by ICP-MS, give a pictures of the different responses performed both to different stress and to combined stress, probably correlated to the up-regulation and/or down regulation of metal uptake proteins. Performed experiments demonstrate that the introgression of the wild genome into the cultivated one produces a new phenotype, perhaps due to the expression of traits linked to uptake, translocation and accumulation of useful and/or toxic metal into plant tissues and organs. Regarding the functional genomics approach for gaining insight into gene networks involved in mineral-ion accumulation in tomato plants, in literature has been reported that at least 25 major family genes are involved for metal homeostasis in plants. Among them, the genes ysl, hma, mtp, znt, zrt have been already studied at least in the plant species Arabidopsis thaliana, A. halleri and Thlaspi caerulescens. So far, no such genes have been reported to be cloned in Solanum species. We have focused our study on the genes YSL1, ZNT1 and MTP1 responsible for uptake, translocation and accumulation of metal such as zinc, cadmium, and iron into plant compartment. For all of them, consensous sequences from nucleotide multialignment have been obtained. Then, each of those were blasted in a Solanum EST collection databank and an assembled UniGene sequence was obtained.. Finally, we have designed primers and performed PCR analysis on S. lycopersicon and S. pennelli genomic DNA. So far, we have cloned a putative ysl1 sequence from tomato, that has shown that a very high percentage of identity (92%) with whole ysl1 gene of Nicotiana tabacum; the in silico translated sequence of this sequence has shown a 89% of identity with the same tobacco protein

Ionome variations in tomato Introgressed Lines (Solanum Pennellii x S. Lycopersicum cv. M82) following metal treatements shed new light on food health.

A tomato introgression line population that combines single chromosomal segments introgressed from the wild, green fruited species Solanum pennelli in the background of the domesticated tomato, S. lycopersicum cv. M82, was used in this study. Results shed light both on the metal accumulation of ILs tomato plants and on theirs ionome modifications

Wastewater phytoremediation: genomic analysis and screening of green microalgae species for extracellular laccase activity.

Phytoremediation deals with the use of plants, or other green photosynthetic organisms, to reduce organic or inorganic pollutant in the environment, mainly waters and soils (Pivetz. B.E. 2001. EPA /540/S-01/500). Our research group is involved in studies regarding the use of plants for soil phytoremediation (Galante et al., 2005. Proc. SIGA Congress, L04) and, more recently, of unicellular green algae species (green microalgae) for wastewater treatments. Some microalgae species have been recently tested to degrade an array of pollutants such as phenols, polyphenolic aromatic compounds (PAH) and even hormones (Pollio et.al., 1994. Phytochemistry, 37:1269- 1272;. Pinto et.al., 2003. Biotechnol Lett., 25:1657-1659). It is worthy to note that about 2,500 species belong from Chlorophyceae, seldom living in contrasting habitat under severe environmental conditions. So far, a little has been done to exploit this genetic biodiversity bonanza; thus, few reports have been published on enzymes implicated in their degradative action (Semple et.al., 1996. Appl. Envr. Micr, 62:1265-1273). Since a wide collection of green microalgae species are available at the University of Naples, Department of Biological Science, recently we have started a research aimed to (a) find algae species with extracellular phenoloxidase enzymatic activity; (b) identify extracellular enzymes able to degrade xenobiotic like synthetic dyes and other PAHs; (c) clone and overexpress genes producing phenoloxidases in homologous and in heterologous systems, in order to use these enzymes primarily for phytoremediation of milling oil wastewaters. Among phenoloxidaes, we focused our interest on laccases (EC 1.10.3.2) that are phenol-oxidoreductases able to catalyze the oxidation of various aromatic compounds (particularly phenols) with the concomitant reduction of oxygen to water. Selected algae strains were grown in liquid culture at 22°C under continuous light conditions, starting with an inoculum of 0.1 OD. After ten days, the algal growth was measured as optical density at 600 nm. A screening was performed by detecting the laccase activity in the broth medium culture, deprived of algae cells, in the presence of 2,2-azino-bis 3-ethybenz-thiazoline-6-sulfonic acid (ABTS) at 420 nm. The laccase activity was referred to the polyphenol oxidase activity of Trametes versicolor; thus, each positive strain was assayed on industrial azo-dye Remazol Brilliant Blue R (RBBR) and on the natural phenol compound syringaldazine by kinetic analysis. Preliminary results, obtained comparing different species, showed a wide variation both within the same substrate and among the different microalgae. Microalgae strains able to produce and secrete laccase enzymes were further chosen for more detailed genetic studies. To clone phenoloxidase genes from those species, we have started a bioinformatics approach, on the basis of highly conserved coding sequences of laccases already isolated and sequenced from several higher plants. Primers drawn on the alignment of those sequences have been used to amplify genomic DNA