JXB Advance Access originally published online on December 13, 2004
Journal of Experimental Botany 2005 56(410):297-307; doi:10.1093/jxb/eri057
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RESEARCH PAPER |
Metabolic profiling of leaves and fruit of wild species tomato: a survey of the Solanum lycopersicum complex
1Department of Lothar Willmitzer, Max-Planck-Institut für Molekular Pflanzenphysiologie, Am Mühlenberg 1, D-14476 Golm, Germany
2The Otto Warburg Center for Biotechnology, Faculty of Agriculture, The Hebrew University of Jerusalem, PO Box 12, Rehovot 76100, Israel
* To whom correspondence should be addressed. Fax: +49 331 5678408. E-mail: fernie{at}mpimp-golm.mpg.de
Received 25 June 2004; Accepted 22 October 2004
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Abstract |
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The domestication of the tomato Solanum lycopersicum and associated selective pressures eventually led to the large-fruited varieties cultivated today. S. lycopersicum varieties are generally red-fruited, but display considerable variance in fruit colour intensity, shape, and quality. The increase in productivity on cultivation is, however, somewhat offset by the narrowing of the crops genetic base which leads to increased susceptibility to biotic and abiotic stresses. Since S. lycopersicum can easily be crossed with its wild species relatives, this exotic germplasm can provide a valuable source for the improvement of agriculturally important traits. A GC-MS based survey is presented here of the relative metabolic levels of leaves and fruit of S. lycopersicum and five wild species of tomato that can be crossed with it (S. pimpinellifolium, S. neorickii, S. chmielewskii, S. habrochaites, and S. pennellii). Changes in metabolite contents were identified in the wild species that are potentially important with respect to stress responses, as well as in metabolites of nutritional importance. The significance of these changes is discussed with respect to the use of the various wild species for metabolic engineering within wide breeding strategies.
Key words: Metabolic engineering, metabolic profiling, plant breeding, Solanum lycopersicum, wild species tomato
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Introduction |
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Fresh-market and processing varieties of tomato both originated from the cherry tomato Solanum lycopersicum ‘cerasiforme’. The domestication of wild cherry types in Mexico spread to Europe and through the process of selection eventually led to the large-fruited varieties (Frary et al., 2000
). Most S. lycopersicum varieties are red-fruited and display considerable variance with respect to fruit colour intensity, shape, quality, growth habit, and leaf morphology (Grandilio et al., 1996; van der Knaap et al., 2002; Frankel et al., 2003; Holtan and Hake, 2003; Yates et al., 2004). However, it is important to note that the increases in productivity conferred by evolution of S. lycopersicum under domestication, like those of all crop species, are somewhat offset by the narrowing of the crop's genetic basis (Zamir, 2001). Whilst exotic germplasm resources often carry many agriculturally undesirable alleles, genetic studies are increasingly being employed in the identification of both undesirable (Tadmor et al., 2002) and agriculturally valuable traits (Rick, 1974; Kovacs et al., 1998; Ray et al., 1999; Sebolt et al., 2000). Once identified, selected traits of exotic resources can be incorporated into commercial elite varieties via introgression breeding (Eshed and Zamir, 1995; Zamir, 2001). In the case of tomato, several examples of the utility of wild species alleles have been realised for some time with several traits being successfully integrated to date. Examples of such integrations include commercial tomato hybrids that contain different combinations of up to 15 wild disease-resistance genes (Pan et al., 2000) and the introduction of important genes from S. pennellii which increase the fruit soluble solids content by 15–25% (Fridman et al., 2000) and provitamin A (ß-carotene) level by more than 15-fold (Ronen et al., 2000).
S. lycopersicum can easily be crossed with a range of other Solanum species including S. pimpinellifolium, S. neorickii, S. habrochaites, S. chmielewskii, and S. pennellii—sometimes collectively referred to as the Solanum lycopersicum complex. These species display markedly different phenotypes from S. lycopersicum most notably with respect to their fruit. All wild species bear fruits that are dramatically smaller than those of the domesticated species and only one of the species are red-fruited. Comparative physiology has been carried out on many of the wild species with particular attention being paid to comparisons of plant performance under different environmental conditions such as aridity (Frankel et al., 2003), high salinity (Monforte et al., 1997a, b; Foolad et al., 2002), and chilling (Venema et al., 1999), as well as susceptibility to biotic stresses such as viral resistance (Legnani et al., 1996). Far less attention, however, has been paid to comparison at the biochemical level with comprehensive surveys essentially restricted to soluble solid contents (Fridman et al., 2000), sugars (Chetelat et al., 1995), and glycoloids (Courtney and Lamberth, 1977). The metabolic profiles of leaves and fruit of S. lycopersicum and five wild species tomatoes that can be crossed with this elite variety (S. pimpinellifolium, S. neorickii, S. habrochaites, S. chmielewskii, and S. pennellii) are reported here. To perform this study all plants were grown alongside each other under carefully controlled growth conditions. Leaf material was harvested 6 h into the light period from the different species after 6 weeks of growth, and fruits were harvested 45 d after flowering. Subsequently these tissues were characterized by utilizing a gas chromatography-mass spectrometry (GC-MS) protocol that has recently been established for tomato tissues (Roessner-Tunali et al., 2003). This method allows the detection and robust quantification of over 90 metabolites of known chemical structure including organic acids, sugars, sugar alcohols, amino acids, and a few soluble secondary metabolites (Fernie, 2003; Stitt and Fernie, 2003; Fernie et al., 2004). Differences in metabolite composition amongst these species will be discussed in the context of their utility for the selection of near isogenic introgression lines for breeding purposes.
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Materials and methods |
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Plant material and growth of plants
Tomato seeds of accession numbers LA3475 (Solanum lycopersicum), LA1589 (S. pimpinellifolium), LA2133 (S. neorickii), LA1028 (S. chmielewskii), LA1777 (S. habrochaites), and LA0716 (S. pennellii) were obtained from the true-breeding monogenic stocks maintained by the Tomato Genetics Stock Centre (University of California, Davis). The seeds were germinated on Murashige and Skoog medium (Murashige and Skoog, 1962
) containing 2% (w/v) sucrose and were grown in a growth chamber at 500 µmol photons m–2 s–1 and 25 °C under a 12/12 h light/dark regime. Experiments were carried out on mature fully expanded source leaves from 6-week-old-plants and on fruits taken 45 d after flowering.
Chemicals
All chemicals were purchased from Sigma-Aldrich Chemie GmbH (Deisenhofen, Germany) with the exception of N-methyl-N-[trimethylsilyl]trifluoroacetamide (Macherey-Nagel GmbH & Co. KG, Düren, Germany).
Starch and protein measurements
Starch and protein were extracted and measured as detailed in Fernie et al. (2001) and Tauberger et al. (2000), respectively.
Extraction, derivatization and analysis of tomato leaf and fruit metabolites using GC-MS
Metabolite analysis by GC-MS was carried out by a method modified from that described by Roessner et al. (2001a). Tomato leaf tissue (250 mg) was homogenized using a ball mill precooled with liquid nitrogen and extracted in 1400 µl of methanol; 60 µl of internal standard (0.2 mg ml–1 ribitol in water) was subsequently added as a quantification standard. The mixture was extracted for 15 min at 70 °C and mixed vigorously with 1 vol. of water. In order to separate polar and non-polar metabolites 750 µl chloroform was then added to the mixtures. After centrifugation at 2200 g the upper methanol/water phase was taken and reduced to dryness in vacuo. For tomato fruit tissue the same procedure was used with the exception that 300 mg of tissue were taken and the extraction mixture was comprised entirely of methanol.
Residues following reduction were redissolved in and derivatized for 90 min at 37 °C (in 40 µl of 20 mg ml–1 methoxyamine hydrochloride in pyridine) followed by a 30 min treatment with 60 µl MSTFA (N-methyl-N-[trimethylsilyl]trifluoroacetamide) at 37 °C. 8 µl of a retention time standard mixture (0.029% (v/v) n-dodecane, n-pentadecane, n-nonadecane, n-docosane, n-octacosane, n-dotracontane, and n-hexatriacontane dissolved in pyridine) was added prior to trimethylsilylation. Sample volumes of 1 µl were then injected onto the GC column using a hot needle technique.
The GC-MS system used comprised an AS 2000 autosampler, a GC 8000 gas chromatograph, and a Voyager quadrupole mass spectrometer (ThermoFinnigan, Manchester, UK). The mass spectrometer was tuned according to the manufacturer's recommendations using tris-(perfluorobutyl)-amine (CF43). Gas chromatography was performed on a 30 m Rtx_5Sil MS column with 0.25 µm film thickness with a 10 m Integra precolumn (Restek, Bad Homburg, Germany). The injection temperature was set at 230 °C, the interface at 250 °C, and the ion source adjusted to 200 °C. Helium was used as the carrier gas at a flow rate of 1 ml min–1. The analysis was performed under the following temperature programme; 5 min isothermal heating at 70 °C, followed by a 5 °C min–1 oven temperature ramp to 350 °C, and a final 5 min heating at 330 °C. The system was then temperature equilibrated for 1 min at 70 °C prior to injection of the next sample. Mass spectra were recorded at 2 scans s–1 with an m/z 50–600 scanning range. Both chromatograms and mass spectra were evaluated using the MASSLAB program (ThermoQuest, Manchester, UK) and the resulting data are prepared and presented as described in Roessner et al. (2001b). The absolute concentrations of most metabolites were determined by comparison with standard calibration curve response ratios of various concentrations of standard substance solutions, including the internal standard ribitol and which were derivatized concomitantly with tissue samples.
Statistical analysis
If two observations are described in the text as different this means that their difference was determined to be statistically significant (P <0.05) by the performance of Student's t-tests.
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Results |
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Experimental design
The plants were all grown simultaneously in a climate-controlled growth chamber under conditions that allowed normal fruit development in all species and which were close to optimal for S. lycopersicum. Material was harvested from equivalent fully expanded source leaves and from fruits 45 d after flowering at which stage the S. lycopersicum fruits were ripe. This time point was chosen for the fruit harvest on the basis of the fact that the metabolite content of S. lycopersicum is relatively constant in the days preceding and following it (F Carrari, AR Fernie; personal communication), and it was therefore reasoned that differences in developmental age between the genotypes would be minimized.
Time after flowering was preferred to other developmental parameters as the criterion for harvesting since it was difficult to choose a suitable parameter for the comparison of such morphologically diverse species. Differences in plant and fruit morphology are illustrated in Fig. 1A and B, respectively (fruits were harvested 45 d after flowering). After 6 weeks and 45 d after flowering, leaf and fruit samples were taken. Tissues were dissected and rapidly snap-frozen in liquid nitrogen. The midrib was removed from leaf material prior to freezing whilst the fruit was skinned and seeds removed and only the pericarp tissue was taken for subsequent metabolite analysis.
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Starch and protein content in fruits of the wild species
As a first experiment the starch and, importantly, the protein content, in samples taken from S. lycopersicum and the wild species were quantified. As would be expected, given the loss of photosynthetic activity on the conversion of chloroplasts to chromoplasts during the ripening process, the green-fruited species contained significantly higher levels of starch than S. lycopersicum and S. pimpinellifolium although only marginally so in the case of S. habrochaites. The protein content was less variable across the species with only S. pimpinellifolium (lower) and S. pennellii (higher) exhibiting significantly different levels of protein in the fruit relative to S. lycopersicum (Fig. 2). Given that the fruit protein content was in approximately the same range between the species, subsequent metabolite data will only be presented here per gram fresh weight (however values expressed per mg protein can be viewed on our webpage: www.mpimp-golm.mpg.de).
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Leaf metabolite contents
Organic acid contents in wild species leaves:
For the purposes of this study calibration curves were generated for every organic acid (and every metabolite for which standards could be obtained—a total of 64 metabolites). As would be expected the major organic acids in leaves of all the species were malate, citrate, succinate, various forms of ascorbate, and glycerate (Table 1). Considerably lower levels were observed in the levels of aconitate (S. habrochaites, S. pimpinellifolium, and S. neorickii), citrate (S. pennellii), and dehydroascorbate (S. habrochaites), galacturonate (S. chmielewskii and S. pennellii), and isocitrate (S. pennellii) with respect to the levels determined for S. lycopersicum. Conversely, significantly greater amounts were observed for citramalate and glycerate (S. chmielewskii and S. habrochaites), glycolate (in all wild species with the exception of S. pennellii), 2-oxoglutarate (S. habrochaites and S. neorickii), maleate (S. neorickii), malate (S. chmielewskii and S. habrochaites), succinate (S. chmielewskii and S. neorickii) and threonate (S. habrochaites) with respect to the amounts determined for S. lycopersicum.
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Sugar and sugar alcohol content in wild species leaves:
The major sugars and sugar alcohols in S. lycopersicum and the wild species were glucose, fructose, sucrose, and inositol as would be expected; with the exception of the sucrose accumulator (S. chmielewskii; Chetelat et al., 1995
) the contents of glucose and fructose are much higher than those of sucrose, most probably due to the high invertase activity present in tomato leaves. Significant decreases were observed in the levels of arabinose (all wild species with the exception of S. pimpinellifolium), fructose (dramatically decreased in all wild species with the exception of S. chmielewskii), fucose (below the level of detection in S. pimpinellifolium and S. pennellii), galactose (below the level of detection in S. neorickii and S. pennellii), glucose (S. chmielewskii and S. habrochaites), maltose (S. pennellii), mannose (in all wild species), mannitol, rhamnose, and ribose (S. neorickii and S. pennellii), xylose (S. chmielewskii, S. habrochaites, and S. pennellii), with respect to the levels determined for S. lycopersicum. Conversely, significant increases were observed in the levels of ribose (S. habrochaites) and sucrose (S. chmielewskii) with respect to the levels determined for S. lycopersicum whereas isomaltose was only above the detection limit in S. neorickii and S. pimpinellifolium and trehalose was only detected in S. chmielewskii and S. habrochaites. The levels of galactitol, gentiobiose (an oligosacharin recently identified in ripening tomato fruit; Dumville and Fry, 2003), glycerol, inositol, maltitol, melizitose, and sorbitol were, however, remarkably similar across the species.
Amino acid contents in wild species leaves:
Leaves from S. lycopersicum are characterized by relatively high contents of aspartate, glutamine, glutamate, oxoproline, proline, threonine, and serine. Significantly lower levels of the following metabolites were observed in leaves from the wild species: 
-aminobutyric acid (GABA), glutamate, and lysine (S. pennellii). By contrast, the levels of the following metabolites were higher in leaves from the wild species: alanine and arginine (S. chmielewskii, S. habrochaites, and S. neorickii), asparagine (S. chmielewskii and S. neorickii), aspartate (S. habrochaites), glutamate, glycine, lysine, and ornothine (S. habrochaites), glutamine and isoleucine (S. chmielewskii), methionine (S. habrochaites and S. neorickii), proline (S. pimpinellifolium), ß-alanine, hydroxyproline, and valine (S. chmielewskii and S. habrochaites), and serine (S. neorickii). The differences observed in absolute values did not, however, always reflect important metabolite ratios. For example, asparagine:aspartate, and glycine:serine ratios are lower in all the wild species. Differences were, however, observed in the ratios of glutamine:glutamate, where ratios are lower in S. lycopersicum (1:0.3), S. pennellii (1:0.6), S. pimpinellifolium (1:0.8), and S. habrochaites (1:0.7), but elevated in S. chmielewskii (1:2.7) and S. neorickii (1:1.5). Despite the fact that the levels of the majority of amino acids were higher in the wild species, levels of leucine, 5-oxoproline, phenylalanine, threonine, tryptophan, and tyrosine were essentially the same in all species.
Miscellaneous metabolite contents in wild species leaves:
In addition, the relative levels of a further 12 metabolites were determined including phosphorylated intermediates, fatty acids, putrescine, and uracil. The hexose glucose 6-phosphate and fructose 6-phosphate were marginally (yet not significantly), lower in the wild species with the exception of S. habrochaites in which they were 3–4-fold higher. Similar trends were also observed in glycerol 1-phosphate and inositol 1-phosphate. The level of 3-phosphoglycerate (3PGA) was significantly higher in S. neorickii, S. pennellii, and S. pimpinellifolium, whilst the level of putrescine was significantly higher in S. neorickii than in S. lycopersicum. Significantly higher levels of shikimate could be observed throughout the wild species. Total phosphate content, fatty acids 16:0 and 18:0, quinate and uracil levels were constant across the species.
Fruit metabolite contents
Organic acid contents in wild species fruits:
The major organic acids of the fruit were somewhat different from those found in the leaf since although citrate and malate and D-isoascorbate were present at high levels, the level of succinate was lower in the fruit (Table 2). Conversely, the levels of galacturonic acid, gluconate, and isocitrate are considerably higher in the fruit than in the leaf. Furthermore, several metabolites such as chlorogenate and nicotinate were only detectable in the fruit.
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Significantly lower levels of the following metabolites were observed in fruits from the wild species when compared on a per gram fresh weight basis to S. lycopersicum (data are additionally expressed per mg protein on our webpage: www.mpimp-golm.mpg.de): dehydroascorbate (S. habrochaites), gluconate (S. habrochaites, S. pennellii, and S. pimpinellifolium), L-ascorbate (S. habrochaites), maleate (S. pennellii), succinate (S. chmielewskii, S. habrochaites, and S. pimpinellifolium), and threonate (S. habrochaites). By contrast, higher levels of the following metabolites were observed in fruits from the wild species than in S. lycopersicum: aconitate (S. chmielewskii, S. habrochaites, and S. pimpinellifolium), citramalate and threonate (S. neorickii, S. pennellii, and S. pimpinellifolium), citrate and dehydroascorbate (all wild species with the exception of S. habrochaites), chlorogenate (massively higher in all lines), fumarate and salicylate (S. chmielewskii, S. neorickii, and S. pennellii.), galacturonate (S. neorickii and S. pimpinellifolium), gluconate (S. neorickii), glycerate (S. chmielewskii and S. neorickii), isocitrate (all species), 2-oxoglutarate (S. pennellii), L-ascorbate (S. pennellii and S. neorickii), malate (massively higher in all species with the exception of S. pennellii), nicotinate (dramatically higher in all wild species), shikimate (all species with the exception of S. pimpinellifolium) and succinate (S. pennellii).
Sugar and sugar alcohol content in wild fruits:
The major sugar and sugar alcohol content of the fruits is very similar to that observed in the leaves with glucose, fructose, sucrose, and inositol again being the major constituents. However, several metabolites such as erythritol and raffinose were observed in the fruit despite being present below the level of detection (if at all) in the tomato leaf. Significantly lower levels of the following metabolites were observed in fruits from the wild species: arabinose (S. habrochaites, S. neorickii, and S. pennellii), fructose and glucose (all species with the exception of S. pimpinellifolium), fucose (S. neorickii and S. pimpinellifolium), galactose (S. pennellii), gentiobiose (not detected in S. chmielewskii or S. neorickii), mannose (S. habrochaites, S. neorickii, and S. pimpinellifolium), raffinose (all with the exception of S. pimpinellifolium which increases massively), ribose (all species) and trehalose (S. habrochaites and S. pennellii). Conversely, higher levels of the following metabolites were observed in fruits from the wild species: fructose and glucose (S. pimpinellifolium), galactose (S. neorickii and S. pimpinellifolium), glycerol and rhamnose (all wild species), inositol, maltose, and xylose (all wild species with the exception of S. pimpinellifolium), isomaltose (S. chmielewskii, S. pennellii, and S. pimpinellifolium), mannose (S. pimpinellifolium) sucrose (S. chmielewskii, S. habrochaites, and S. neorickii). The levels of erythritol, maltitol, mannitol, sorbitol, and galactitol were observed to be invariant across the species.
Amino acid contents in wild species fruits:
The pattern of amino acids of the fruits was largely similar to that of the leaves with the same amino acids being major constituents of each tissue. However, the levels of GABA and the derivatives of proline were notably higher in the fruit than in the leaves. In sharp contrast to the situation observed in the leaves, the majority of amino acids were found at lower quantity in the wild species than in S. lycopersicum with significant differences being observed in the following metabolites: arginine, asparagine, isoleucine, leucine, and tyrosine (in all wild species with the exception of S. habrochaites), aspartate (S. habrochaites, S. pennellii, and S. pimpinellifolium), ß-alanine, GABA, and methionine (in all wild species with the exception of S. pennellii), glutamine, glutamate, lysine, phenylalanine, serine, hydroxyproline, and threonine (in all wild species, dramatically so in the cases of glutamine and glutamate), glycine (all wild species with the exception of S. neorickii), ornithine and oxoproline (all wild species with the exception of S. habrochaites), proline (S. pennellii and S. pimpinellifolium), tyrosine (S. neorickii, S. pennellii, and S. pimpinellifolium) and valine (S. chmielewskii, S. neorickii, and S. pimpinellifolium). The exception to this trend was the fact that alanine, tryptophan, and valine were all present at significantly higher levels in S. habrochaites than in S. lycopersicum. Studying important metabolite ratios of the fruits revealed differences across the species. In the case of the asparagine:aspartate ratio S. chmielewskii, S. neorickii, and S. pimpinellifolium display much lower ratios, whereas S. habrochaites and S. pennellii display much higher ratios than those observed for S. lycopersicum. A similar picture emerges in the case of glutamine:glutamate with the exception that the ratio in S. pennellii is not significantly different from S. lycopersicum. Conversely, the glycine:serine ratio was much lower in all wild species with the exception of S. habrochaites.
Miscellaneous metabolite contents in wild species fruits:
The relative levels of a further 18 metabolites in addition were determined including the same miscellaneous metabolites detected in the leaves in addition to 
-tocopherol, spermidine, tyramine, fatty acid 18:2, and dopamine. The hexose phosphates glucose 6-phosphate and fructose 6-phosphate were significantly lower in S. pennellii, and glycerol 1-phosphate was lower in S. pennellii and S. pimpinellifolium than in the cultivated tomato, but glycerol 1-phosphate levels were significantly higher in S. habrochaites (than in the cultivated tomato). S. chmielewskii, S. habrochaites, and S. neorickii all displayed higher levels of inositol 1-phosphate than S. lycopersicum. The contents of free phosphate and fatty acid 16:0, 18:0, and 18:2 were higher in all of the wild species with the exception of S. pimpinellifolium. By contrast, the levels of -tocopherol were lower in all the wild species, but only marginally so in the case of S. chmielewskii. Changes were also observed in dopamine (which was higher in S. chmielewskii and S. neorickii, but was not detected in S. pennellii and S. pimpinellifolium), putrescine (which was lower in S. chmielewskii, S. neorickii, and S. pennellii), tyramine (massively increased in S. pimpinellifolium), and uracil (which was higher in S. chmielewskii, S. neorickii, and S. pennellii).
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Discussion |
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This study provides the first comprehensive comparative analysis of the metabolite composition of leaves and fruits from the elite tomato species S. lycopersicum and five wild species tomatoes (S. pimpinellifolium, S. neorickii, S. chmielewskii, S. habrochaites, and S. pennellii). Despite the fact that many of the wild species are green-fruited it is difficult to gauge developmental equivalence. Therefore, for the purposes of this study all fruits were harvested 45 d after flowering. The techniques described in this paper were previously used to phenotypically characterize genetically distinct potato and tomato lines exhibiting altered sucrose breakdown and hexose phosphorylation, respectively (Roessner et al., 2001a
; b; Roessner-Tunali et al., 2003; Carrari et al., 2003). During the course of these earlier studies the GC-MS method was optimized to allow the evaluation of the relative levels of over 60 metabolites in the leaf and fruit of tomato, and furthermore established that there were large differences in a broad range of metabolites during fruit development (Roessner-Tunali et al., 2003). The number of metabolites evaluated in both the fruit and leaves was extended to 82 and 71 compounds, respectively (largely by the procurement of additional chemical standards), and furthermore evaluated the absolute concentrations of the vast majority of these compounds. In addition, the levels of starch and protein in fruit samples were also evaluated. The latter measurements were carried out in order to assess the influence of water content on the changes observed in metabolite levels. There is a tremendous variance in metabolite content in both leaves and fruits of the wild species. On the basis of leaf metabolite content S. pimpinellifolium is the closest wild species to S. lycopersicum (in that it shows the least significant differences) followed by S. pennellii, S. chmielewskii, and S. neorickii whilst S. habrochaites is the most distinct. This may have some basis in the botanical relationship of the species with S. pimpinellifolium being the only red-fruited wild species and the only one to have exhibited a natural introgression with S. lycopersicum. In fact, it is most probable that both species evolved from a common ancestor. S. chmielewskii, S. neorickii, and S. pennellii are all green-fruited species native to Peru. The first two favour growth in moist conditions whilst the latter evolved in hot dry environments and has long been regarded as a good source for drought resistance and insect tolerance genes. S. habrochaites, native to Southern Ecuador is a trichromous green-fruited species with tolerance to low temperature and has been noted to be resistant to several pests as well as containing a high concentration of the naturally occurring pesticide 2-tridecanone. The general pattern of metabolite content in the fruits is, however, somewhat reversed with S. habrochaites showing the fewest significant changes compared with S. lycopersicum. followed by S. chmielewskii, S. pennellii, S. pimpinellifolium, and finally, S. neorickii. It should be noted that, with the exception of S. habrochaites, all the wild species showed substantially higher levels of metabolic variation in fruits than in the leaves—a fact that probably could be expected to be due to the higher degree of morphological variance in these organs.
The fact that all the wild species studied here can interbreed with S. lycopersicum has long been exploited by plant breeders with particular attention being paid to biotic and abiotic stresses (Montforte et al., 1997a, b; Venema et al., 1999; Pan et al., 2000). Clear links between resistance to these phenomena and metabolite composition have been demonstrated. For example, biotic resistance is often conferred by the synthesis of molecular repellents (Mitchell-Olds et al., 1998) whereas proline has been reported to play an important role in water stress (Brugiere et al., 1999) and several solutes, especially hexoses have frequently been implicated in response to cold stress (Gilmour et al., 2000). Whilst the data presented here may ultimately be of great use in selecting breeding material for the improvement of the above-mentioned traits, the biochemical basis underlying them is currently unclear. Furthermore, it is likely that such complex traits will be influenced by a wide range of genes and/or biochemical interactions. Another long-term aim of plant breeders over many years has been in fruit quality improvement (Saliba-Colombani et al., 2001; Lecomte et al., 2004). Substantial progress has been made in this field and the metabolic basis of several quality traits has been established. Brix (the total soluble sugars content, mainly constituting hexoses, citrate, and malate; Fridman et al., 2000), organoleptic properties, and analysis of volatiles (Saliba-Colombani et al., 2001) have all been examined in wide crosses between wild species and the cultivated S. lycopersicum. Quantitative trait loci (QTL) for physical and chemical traits have been established (Fridman et al., 2000; Saliba-Colombani et al., 2001). In the case of Brix 9-2-5 a QTL for soluble sugar content has been delineated to a single nucleotide of the apoplastic invertase LIN5 (Fridman et al., 2004). The wide metabolic variance of primary metabolites in fruits of the wild species suggests that similar approaches aimed at boosting the levels of nutritionally important metabolites such as lysine, methionine, ascorbate, and tocopherol will stand a high chance of success. The dramatically higher levels of secondary metabolites that it was possible to detect by GC-MS also suggests that these wild species represent a valuable resource both for the increase of flavour compounds such as volatiles (Saliba-Colombani et al., 2001; Bovy et al., 2002) and carotenoids (Liu et al., 2003) and for the natural product chemist (Lamartiniere, 2000; Dixon and Sumner, 2003). Rich resources of exotic libraries in which marker-defined chromosome segments from the wild species have been introgressed into the cultivated variety have already been established (Paterson et al., 1990; Eshed and Zamir, 1994). These will undoubtedly represent an import tool for metabolic engineering, particularly given the current problems of consumer acceptance of products modified by transgenesis.
In conclusion, in recent years there has been an increasing interest in analysing various biological properties of natural genetic diversity (Maloof, 2003; Koornneef et al., 2004). This study provides a compendium of metabolite levels from leaves and fruits of S. lycopersicum and wild species tomato. In combination with prior botanical and genetic studies on adaptability to diverse climates and resistance to biotic stress, these data provide correlative information that may, with further experimentation, allow the elucidation of biochemical factors underlying these phenomena. Furthermore, they provide information that may be of considerable importance for breeding-driven metabolic engineering of nutritionally important metabolites.
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Acknowledgements |
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We are grateful to Britta Hausmann for excellent care of the tomato plants and to Lothar Willmitzer for discussions and support over the last years. We greatly appreciate Sandra Knapp's (Natural History Museum, London, UK) effort to provide us with the new Solanum nomenclature.
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Footnotes |
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Abbreviations: GC-MS, gas-chromatography-mass spectrometry; GABA,
-aminobutyric acid; 3PGA, 3-phosphoglycerate; DAF, days after flowering; FW, fresh weight; LYC, Solanum lycopersicum; PIM, Solanum pimpinellifolium; NEO, L. neorickii; CHM, S. chmielewskii; HAB, S. habrochaites; PEN, S. pennellii. |
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