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Tomato, or Solanum lycopersicum is a red, edible fruit which originates from South America, and spread around the world postdating the Spanish Colonization of the Americas. It is now widely grown in temperate countries with cooler climates. Now, it is one the most widely grown fruit and consumed in the world, having more than 122 million tons being produced worldwide in 2005 (FOASTAT, 2005). Tomato is part of the Solanum family which contains many other plant species of commercial and/or nutritional interest (e.g. potato, pepper, eggplant, tobacco, and petunia). Being colligated with several quality attributes such as high nutritional value like antioxidants, together with ranges of flavour volatiles, flavanoids, vitamins, caretenoids, all of which are of greatly pertinence to the market consumption and demand. In addition, tomato fruit is a important natural source of lycopene, a caretenoid, which has been the subject of increasing interest due to its health beneficial effect, particularly on prostate cancer prevention (Basu and Imrhan, 2007; Jatoi et al., 2007).
Tomato and Secondary Metabolites
Besides its proposed nutritional values, tomato represents a model and is widely used for studying various type of fruits. During ripening, fruits will undergo many series of biochemical changes such as alterations in pigments biosynthesis and volatile productions (Brady, 1987), couple with the softening of cell wall resulting from the cell wall components (cellulose, hemicellulose, & pectin) modifications, increased susceptibility to pathogen resistance, increase in ascorbic acid and total soluble solids content (Fraser et al., 1994; Giovannoni, 2001; Carrari et al., 2006). In climacteric fruits, such as tomato fruit, ethylene plays a major role in fruit development and ripening, in addition to other plant hormones such as auxin and abscisic acid, as well as gibberellins and cytokinins (Srivastava and Handa, 2005). Little is known between the interaction of ethylene and auxins during fruit growth and ripening in some species as for example kiwifruit (Actinidia deliciosa). A synthetic auxin, 3,5,6-trichloro-2-pyridyloxyacetic acid (3,5,6-TPA), in action with ethylene is able to increase fruit size Japanese plum (Prunus salicina Lindl.) (Raphael et al., 2007). On the other hand, in tomato the interactions between hormone and metabolites synthesis was shown to be relative. Elsadig & James, 1984 concluded that accumulation of tomatine during the early stages of fruit development must be due entirely to synthesis. Metabolites and hormones may working together coherently throughout the development stages of fruits (Stephen, 2008). In addition, the dynamics and interactions within fruit metabolic pathways, as well as the identity and concentrations of the interacting metabolites during fruit development, are mostly unknown. This include various tomato cultivars which may possibly response differently and produce dissimilar percentage of metabolite groups under biotic or abiotic stress. Metabolites accumulation varies on different parts of the plant too. Biosynthetic genes are responsible for the formation of secondary metabolites such as acids, ï¬‚avonoids and terpenoids may be highly expressed in such tissues where the metabolites are majorly accumulated, whilst these natural compounds translocate among plant organs as well, for example biosynthetic genes such as nicotine, a pyrrolidine alkalaoid of Nicotina species, are found accumulated in leaves (sink organ) even though it is mostly expressed in root tissues (source organ) (Shoji et al. 2000).
In addition, the metabolite composition of fresh tomatoes can vary between the tissues of a single fruit too (Moco et al., 2007; Mounet et al., 2007; Peng et al., 2008) and between different tomatoes, according to the cultivar in question, its cultivation conditions, such as light, temperature, soil, fertilisation and ripeness at harvest, and handling and storage methods (Davies and Hobson, 1981; Dorais et al., 2008; Dumas et al., 2003; Gautier et al., 2008; Schindler et al., 2005; Slimestad and Verheul, 2009).
Thus, diagnosing plant condition can be facilitate with metabolomics by having a direct relationship to the exhibited visual characteristics (phenotype). The chemical diversity of the metabolites is enormous in addition to a large dynamic concentration range. A wide variety of methods have been used to separate and quantify components of the metabolome, and no single analytical platform can capture all metabolites in one sample. For that reason only a technology platform consisting of several approaches based on different techniques offers a solution today. Â The best choice of platform for the study depends heavily on the findings in the interest. Nowadays roughly two different strategies can be established for metabolite investigations: 1)metabolic profiling and 2)metabolic fingerprinting (Dettmer et al., 2007).
Metabolic profiling is the study of metabolites of interest, defined as a priori (e.g. fatty acids, oxidized lipids, nucleosides etc.) and all these metabolites are incisively quantified. Metabolic profiling is a directed way to study different aspects of metabolism and it requires the aggregation of a whole suite of quantitative methods to turn metabolic profiling into metabolomics (Dettmer et al., 2007). In general, metabolic fingerprinting refers to metabolite profiles are acquired and compared with a priori knowledge of the metabolites of interest. Semi quantitative data are acquired by high throughput generic analytical methods (such as LC-MS or 1H-NMR) and (bio)markers (ions or chemical shift signals) are revealed by multivariate statistical tools. The identity of the signals of interest from the fingerprint can subsequently be revealed by metabolite identification procedures.
By using metabolomics technologies, a comprehensive explanation of naturally-occurring metabolites (primary and secondary metabolites) in a biological system, such as tomato fruit, has become practicable. The recent expansion of metabolomic technologies has resulted in the broader use of a diverse range and configuration of instruments and analytical methods. Mostly MS (Schauer et al., 2005; Tikunov et al., 2005; van der Werf et al., 2005; Moco et al., 2006; Fraser et al., 2007) and NMR (Keun et al., 2002; Le Gall et al., 2003; Ward et al., 2003; Kochhar et al., 2006; Griffin and Kauppinen, 2007) technologies are used, but also other techniques such as LC-photo-diode array (PDA) (Porter et al., 2006), infrared and Raman spectroscopy (Ellis and Goodacre, 2006) have been used for plant metabolomics. Among a wide variety of applications (Hall, 2006; Schauer and Fernie, 2006), plant metabolomics approaches are providing insight into the biochemical fingerprints that specific cellular processes in the plant system leave behind, allowing the validation of links to possible metabolite functions.
Metabolites are grouped into primary and secondary, though the definitions between these groups can sometimes be coalesced (Hounsome et al., 2008). Primary metabolites, such as organic acids, fatty acids, nucleotides and amino acids, play essential roles in growth and development, respiration and photosynthesis and hormone and protein synthesis. Secondary metabolites, including phenolic acids, ï¬‚avonoids and terpenoids, play key roles in protecting plants from herbivores, microorganisms and UV radiation, in attracting pollinators or seed-dispersing animals and acting as stress-condition signalling molecules, among other important functions (Crozier et al., 2006).
Within the numerosity of metabolites that constitutes the tomato fruit metabolome, carotenoids, ï¬‚avonoids, phenolic acids, and alkaloids can be analysed using LC-MS techniques. A variety of biological functions have been assigned to these classes of secondary metabolite, for example, as components involved in pollination, photoprotection, seed dispersal, adaptation to abiotic conditions, and defence, as well as being involved in other non-ecological phenomena such as auxin transport, and ethylene signal transduction effect on tomato (Giovannoni, 2001; Friedman, 2002; Taylor and Grotewold, 2005; Kunz et al., 2006). Furthermore, biochemical studies on crops, including tomato fruit, may generate knowledge that potentially can have a direct consumer impact as it provides insight into nutritional and quality aspects.
Cytochrome P450 Enzymes and Secondary Metabolites
On the contrary, cytochrome P450 enzymes (CYP450s) in biosyntheses of some plant secondary metabolites. In the present review, cytochrome P450 monooxygenases involved in the biosyntheses of three structurally and biosynthetically interesting compounds, secologanin, cornoside, and shikonin (Yakugaku, 2005). This is not surprising as nearly every aspect of plant biology is highly dependent on cytochrome P450 enzymes (Choe et al.1998). In addition, it was also known to being essential for both plant steroid hormone biosynthesis and inactivation (Nomura & Bishop 2006). The most basic and crucial involvements of CYP enzymes are particularly on the plant development in the early postembryonic stage (Szekeres et. al 1996), and triggering plant defence mechanisms against pathogens ((Hoagland, 2009, Pegg, 1986). Losses of horticultural produce are a major problem in the post-harvest chain. They can be caused by a wide variety of factors, from attack of diseases, pathogens to the faulty in the processing chain (World Resources Institute, 1998). Diseases are an important source of postharvest loss depending on season, region and handling practices. Commonly, decay or surface lesions peculiarly on tomato resulted from the fungal pathogens Alternaria (Black Mold Rot)(Davis, 1997) , Botrytis (Gray Mold Rot) (Badawy, 2009), Geotrichum (Sour Rot) (Sharma et al., 2006), and Rhizopus (Hairy Rot) (Stevens, 2004). Bacterial Soft Rot caused by Erwinia spp. (Expert, 1999) can be a serious problem particularly if proper harvest and packinghouse sanitation is not used.
Previous report (Noordermeer, 2001) stated, as plants ceaselessly have to defend themselves against life-threatening especially against potential pathogens, it was discovered however, the lipoxygenase pathway plays an important role which the products of this pathway are involved in wound healing, pest resistance, and signalling, or they have antimicrobial and antifungal activity. Interestingly, it has been discovered that hydroperoxide lyase (HPL) which belongs to a special class of cytochrome P450 (Matsui et al. 1996) enzymes cleave the lipoxygenase pathway's products into forming important phytooxylipin which contributes to wound healing, antifungal, and antimicrobial activity signalling. The HPL gene from Arabidopsis thaliana contains a chloroplast-directing transit sequence, (Bate et al. 1998) but the genes from tomato and alfalfa do not (Howe et al. 2000, Noordermeer et al. 2000, Matsui et al. 2000). The intracellular localization thus remains unclear and should be further studied by immunocytochemical methods.
On the contrary, Î±-tomatine was found toxicity and inhibit the development of weeds, crops and phytopathogenetic fungi (Hoagland, 2009). Recently, Cohen et al. 1993, reported that jasmonic acid (JA) and its methylester induces resistance in tomato towards Phytophthora infestans. Then again, Sandrock et al. demonstrated that Phytophthora infestans shows inherent intolerancy towards Î±-tomatine. Î±-Tomatine is a steroidal glycoalkaloid present in Solanaceous plants, principally in a number of Lycopersicon and Solanum species. High concentrations are found in leaves, stems, rootsand green fruit of tomato plants, suggesting a possible role in resistance to pathogens. The toxic effects of Î±-tomatine are attributed to its ability to complex with membrane sterols, causing pore formation and leakage of cell contents. Pathogenic fungi to tomato are usually less sensitive to tomatine than pathogens of other plant species and saprophytes (Rubio et al. 2001). However, there are very little study performed with supporting data of the potential suspect of CYP450s behind the production of Î±-tomatine and the possible elevated expression or repression by the gene encoding for the CYP450 enzymes, on different parts of the tomato plants particularly the leaves and the fruit.
Agrobacterium-mediated transient assays of gene expression in Tomato
The need for quick and simple analyses of gene function has increased since the identification of thousands of sequences through genome projects. It involves one or more transgenic approaches to express or silence a gene. However, the transformation and regeneration of most higher plants remain tedious, time-consuming and often costly. Even with species for which these procedures have been greatly simplified, such as Arabidopsis thaliana or Medicago truncatula (Bechtold et al., 1993; Clough and Bent, 1998; Trieu et al., 2000), it can still take several months to produce transgenic plants suitable for analysis. In addition, because the level of transgene expression can vary from one transgenic plant to another as a result of gene silencing phenomena or the gene's position in the chromatin (reviewed by Vaucheret et al., 1998), multiple transgenic individuals are required for the reliable analysis of a single transgene.
Transient assays mediated by agroinfection have been increasingly employed as an alternative to the analysis of stable transformants. Transient expression provides a rapid method for assaying the function of some types of gene; transgenes can often be assayed within a few days of infiltration (Janssen and Gardner, 1989).
Agrobacterium-mediated transient assays have some limitations, however. They are restricted to species and tissues that are biologically compatible and physically accessible to A. tumefaciens In addition, because two living organisms participate in the process, the efficiency of transient assays is influenced by experimental variables that affect the virulence of A. tumefaciens and the plant's physiological condition. The efficiency of transformation is greatly influenced by the compatibility between plant and bacterium. Some strains of Agrobacterium are more virulent than others on a particular plant species and, conversely, some plant species or genotypes are more or less sensitive to particular strains of Agrobacterium (De Cleene and De Ley, 1976; Anderson and Moore, 1979).
Most laboratory strains of A. tumefaciens elicit necrosis in tomato (Van der Hoorn et al., 2000; and Tadeusz et al., 2005). Sufficient numbers of infected and transformed cells can regenerate into transgenic tomato plants, and therefore the necrotic response has not prevented the utilization of A. tumefaciens for the generation of stable transformants of tomato. However, necrosis can be problematic for Agrobacterium-mediated transient assays of Solanaceous species. Tadeusz et al., 2005 reported that the laboratory Agrobacterium tumefaciens strain C58C1 was the best strain for use in plant species that did not elicit a necrotic response to A. tumefaciens and a wild A. tumefaciens strain, 1D1246, was identified that provided high levels of transient expression in solanaceous plants without background necrosis, enabling routine transient assays in these species.
Take together, this research will be conducted in three fold, (i) Metabolic profiling, which will be focusing on phenolic compounds in methanol extracts of tomato fruit at different developmental stages and leaves, (ii) metabolic fingerprinting, where tomato fruits and leaves metabolite profiles are acquired and compared with the metabolites of interest, especially Î±-tomatine., (iii) Agrobacterium-mediated transient assays of CYP450 gene expression in tomato and will screened for both metabolic profiling, and fingerprinting.