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

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

Green microalgae and plants expressing fungal laccases useful for enviromental and industrial applications

A biotechnological approach to enhance the efficiency of xenobiotic removal from green organisms is to overexpress genes involved in metabolism, uptake or transport of specific organic pollutants. However, a useful approach is to secrete laccases in the soil or water. The laccase cDNA of the poxA1b gene from Pleurotus ostreatus, carrying a signal peptide sequence for enzyme secretion and driven by the CaMV 35S promoter, was cloned into a plant expression vector. Nuclear genetic transformation was carried out by co-cultivation of Agrobacterium tumefaciens with tobacco cv Samsun NN leaves and cells of five different microalgae accessions belonging to the genera Chlamydomonas, Chlorella and Ankistrodesmus. Transgenic plants and microalgae were able to express and secrete the recombinant laccase in the root exudates and the culture medium, respectively. In comparison to untransformed controls, the ability to reduce phenol content in OMW solution was enhanced up to 2.8-fold in transgenic tobacco lines and by up to about 40% in two microalgae accessions. The present work provides new evidence for metabolic improvement of green organisms through the transgenic approach useful for environmental and industrial applications

A NEW RESTORER OF FERTILITY GENE ALLELE FOUND IN PETUNIA x HYBRIDA

In a frame of a project funded by MiPAAF, on selected petunia lines (parental lines, their F1 and their "refreshing" line) DNA sequences specific to male-sterility and restorer genes have been identified and cloned. The presence of sequences from a petunia restorer gene was identified in all genotypes. Hence, the related amplicons were sequenced and they resulted in a never-described before allele. The mutation found can be used for cultivar protection