Molecular Profiling-Fruit Carotenoids Components of Six American Heirloom Tomatoes (Solanum lycopersicum)

Fruit pigments of six vine-ripening American heirloom tomatoes (Solanum lycopersicum) were analyzed: the green-ripe ‘Aunt Ruby’s German Green’, the red-ripe ‘Black from Tula’, ‘Cherokee Purple’ and ‘German Johnson Regular Leaf’ and the yellow-ripe ‘Kellogg’s Breakfast’ and ‘Yellow Brandywine Platfoot Strain’ which were grown in Hungary (Godollo). In total, twenty-one type of pigments were determined by Reverse Phase (RP) HighPerformance Liquid Chromatography (HPLC): the orange colorations of lutein, β-carotene, β-cryptoxanthin, mutatoxanthin and neoxanthin, the red-orange colorations of lycopene, lycopene-epoxide 1, lycopene-epoxide 2, lycoxanthin, 9-cis-lycopene, 13-cis-lycopene, lycopene-diepoxide 1 and lycopene-diepoxide 2 and the third group of colorations of violaxanthin, neochrome, prolycopene, neurosporene-epoxide, neurosporene, ζ(Zeta)-carotene, ζcarotene-like, and α(alfa)-cryptoxanthin. Tomato ‘Black from Tula’ showed the highest content of β-carotene (23.56 g kg-1). The highest lycopene content (19.25 g kg-1) was found in the ‘Cherokee Purple’ and an extremely high prolycopene (syn.: tetra-cis-lycopene or all-trans-lycopene) content was found in the two yellow fruited tomatoes of ‘Kellogg’s Breakfast’ and ‘Yellow Brandywine Platfoot Strain’ (100.87 and 70. 99 g kg-1, respectively). Brix indexes did not show significant differences. Based on the results suggestions for growing purposes and further use in metabolomics and molecular and DNA profiling are given.

Color of tomato fruit skin and flesh is one of the most important quality components of the tomato in the market. The main acyclic carotenoids of ζ-carotene (its color is light-yellow), neurosporine (yellow-orange), lycopene (red-orange), cyclic-carotenoids of γcarotenes (pink-orange) and β-carotene (orange) are the main tomato fruit colorant, and slightly depended on the elution solvents used for the analyses [13,14]. The amount of β-carotene, the main orange colorant and lycopene which causes red-to-orange coloration, are the predominant tomato pigments [15]. Chlorophylls, the green pigments of unripe fruits, breaks down during ripening, except in cultivars with 'black' colored fruits, where persistent chlorophyll content gives purplish-brown color together with the red-to-orange colorations. Persistent chlorophyll content is regulated by gf (green flesh) gene, that encodes a Stay-Green senescence-related regulator, and it finally results in the mix of red and green color, which seems black/brown/chocolate color [16].
Functionally, carotenoids, especially β-carotene, primarily act as accessory and photoprotective pigments for chlorophyll a and b of LHCs (Light Hirvesting Comlex) of photosystem I and II (PSI and PSII) during photosynthesis [17] in leaves, fruit skins and flowers. They absorb the sunlight in a broader range of the blue spectrum (400-500 nm) than chlorophylls, and they transfer this absorbed extra energy to chlorophyll a of the photosynthetic reaction center. Carotenoids also supply substrates for the biosynthesis of the plant growth regulator abscisic acid (ABA) [18]. All carotenoids can became crystallized in the chromoplasts during the transition of chloroplast to chromoplast, or transported and accumulated in lipid bodies.
In animals, ceto-carotenoid type astaxanthin is responsible for the orange color of salmon meat and lobster shell. Feather colors of the birds also came from carotenoids [19]. Chicken egg yolks are rich in lutein and zeaxanthin [20]. In human nutrition and health, carotenoids act as anti-aging and anti-cancer substances and provide provitamin-A (e.g. β-carotene, β-cryptoxanthin and α-carotene) [10]. Wide ranges of carotenoids of algae, fungi and bacteria have also been identified and characterized [21].
Genes of enzymes involved in carotenoids synthesis are encoded in plant nuclear genomes and gene products are transported either to the cytoplasm (including mitochondria) (i.e. mevalonate pathway) or to the plastids (i.e. non-mevalonate pathway) where they are posttranslationally modified and activated [17].
Tomato fruits are also rich in phenols and polyphenols (like gallic acid, catechin, rutin, ferulic acid etc.) and vitamin E (α and γtocopherol), which are also responsible for the antioxidant capacity of the soluble phase of fruit sap [22].
The aim of the study presented was to determine the carotenoids content of six American heirlooms for pigment compositions and to describe the differences with the aim of utilizing the information for future breeding purposes. Tomato plants were staked with three foot bamboo stakes attached with twist ties. All suckers below the first flower cluster were removed in accordance with Kemble et al. [23]. Treatments were randomly assigned to six individual plants in a completely randomized design with three replications.

Plant materials
Data gathered included germination vigor and fruit quality characteristics. The first six ripened fruit grown on the same vine nodes were collected and processed for HPLC and Brix analyses according to Pek et al. [24] and Daood et al. [25].

Extraction of carotenoids
Lipids and fat soluble pigments collected from the raw tomatoes were extracted according to Abushita et al. [22] with slight modifications. Five-gram samples of tomato fruits were taken from each variety in four replicates (6 varieties x 4 replicates = 24 samples) and grind in a crucible mortar with quartz sand followed by adding 20 mL cc. methanol. The mixture was then transferred quantitatively to a 100 mL conical flask and 70 mL of a 6:1 dichloroethane:methanol solution was added. The mixture was shaken for 15 min by a mechanical shaker till the dichloroethane phase was clearly separated from the polar phase (water + methanol). The two phases were separated and the lower layer containing lipids dissolved in dichloroethane was dried over anhydrous sodium sulphate. Finally, the organic solvent was evaporated under vacuum by rotary evaporator at 40˚C. The residues were re-dissolved in HPLC-grade acetone before injection onto HPLC column [25].
HPLC peak identification was based on the comparison of spectral properties and retention time of carotenoids separated with those of available molecular standards of lycopene, β-carotene and zeaxanthin (Sigma-Aldrich, Budapest, Hungary). In case of carotenoids with no available standards, the peaks were identified according to their spectral characteristics and chromatographic retention according to Ritter and Purcell [26] Liaaen-Jensend and Lutences [27] and Borsarelli and Mercadante [28]. The cis isomers of lycopene were identified on the basis of appearance of an extra absorption wavelength at 340 nm and 361 nm. The 9-Z-and 13-Z cis lycopene isomers were identified according to the II-value, which equals to absorbance at 361 nm over absorbance at the maximum wavelength according to Liaaen-Jensend and Lutences [27]. The column effluents were detected and integrated at their maximum absorption wavelength for quantitative determinations and were quantified as either lycopene-or β-carotene equivalents (μg g-1 equal to g kg-1) according to their spectral characteristics according to Rodriguez-Amaya [13].

Statistical analysis
Mean values of four (n=4) HPLC measurements and 95 % Confidence Interval for Mean (CI95% = x ± d) confidence intervals were calculated at P 95% confidence level by SPSS program package. For dendrogram (using Average Linkage Within Group) and Canonical Discriminant Functions Analyses the SPSS program package was also used.
All the other tomatoes showed some radial crack resistance, which is controlled by recessive alleles of cr and ra however 'Black from Tula' [2] was found susceptible to fruit bursting, which is regulated by dominant gene BT (burst types) [29].
The first three American heirlooms [1][2][3] were found to carry Abg (Aubergine) gene, which cause purple fruit epidermis particularly on shoulder, and the 'Yellow Brandywine Platfoot Strain' [6] was found to probably carry fs (fruit stripe) gene, which causes dark green radial stripes at the opposite locules ( Figure 2) (Table 1).   Genetically, the synthesis of plant carotenoids links to the processes of fruit ripening (i.e. cell wall softening), which is regulated about 50 genes in tomato [1]. Regulation/modulation of fruit ripening of all fleshy fruit plant species has profound agronomic importance. Recently, fresh market tomatoes include only 'long shelf-life' varieties, which are natural mutants, and carry ripening inhibitor (RIN) gene(s). The main RIN genes are the rin (ripening-inhibitor MADS-box gene) nor (non-ripening transcription factor gene) nr (never-ripe ethylene signaling) nr-2 (never-ripe 2) / gr (green-ripe) and cnr (colorless nonripening) [30,31]. Of them, one of the earliest tomato fruit ripening mutants was the dominant NR (Never-ripe) mutation [32]. This mutation was shown to be the consequence of a single amino acid change in one of the seven ethylene receptors (LeTR1-7) [23]. The first registered RIN tomato, the 'Daniela'(FA144) an indeterminate longself life hybrid, was released in about 1992 by the BonTom Tomato Breeding Group (Faculty of Agriculture, Hebrew University of Jerusalem, Israel) [3]. Green-Ripe (GR) and its allele, NR-2, were also found as a dominant nonripening mutation [31][32][33][34][35]. One of the other unique natural tomato mutants is a dwarf type 'Micro-Tom' [36] with obviously small fruits.
In our work presented, none of the studied heirlooms showed the presence of any RIN genes by visual observation (Figure 2), as the fruits were ripened and softened very quickly (in some days immediately after the total fruit size development). These fruit characters obviously suggest the marketing of these heirlooms for fresh consumption and tinned juice production.
All heirlooms also showed 'indeterminate' growing habit. Nearly a century ago, a spontaneous mutation in SP (self-pruning) gene family spawned the 'determinate' tomato development which now dominate the tomato market being beneficial for mechanical harvesting [1].
Pigments characteristics: Fruit pigments compositions of the heirlooms showed three main groups ( Figures 3) and (Figure 4) ( Table 2). In the irst group, on the contrary of the extremely low carotenoids compositions ( Table 2) ' Aunt Ruby's German Green' [1] was found delicious taste due probably to its tasty compositions of other organic fruit components of carbohydrates and organic acids [39].

Formulas
As the worldwide monoculture of tomatoes (i.e. less then ten cultivars are cultivated in the World), these unique heirlooms may provide basal material for feeding experiments and medical studies. e.g., eggs of carotenoid-fed female birds (Larus fuscus) were found with high carotenoid contents but low Ig immune globulins (i.e. passive immunity). Whereas, control females produced eggs containing low carotenoid but high Ig content, which results indicated a carotenoid-mediated effects of phenotypes for ecological fitness of mother birds and their offspring [8]. Carotenoids of lutein and zeaxanthin supplemented in male zebra finch birds (Taeniopygia guttata) showed elevated blood carotenoid levels with increased cell mediated and humoral immune responses than control birds, which were coupled with brighter beak coloration, which suggested that carotenoids-based colour signals in birds may directly signal male health via the immunostimulatory action of ingested carotenoid pigments [28]. In depletion of carotenoids of nineteen healthy adult people who were fed at controlled low carotenoids diets for 10 weeks, of the six major human serum carotenoids of lycopene, β-carotene, αcarotene, lutein, zeaxanthin and β-cryptoxanthin, the lycopene concentration showed sharp decrease compared to other carotenoids [12]. This reference indicated that lycopene appears to be the physiologically most important antioxidant of human body [13].

Conclusion
In conclusion, as a result of high prolycopene and ζ-carotene contents, the two yellow fruited heirloom tomatoes 'Kellogg's Breakfast' [5] and 'Yellow Brandywine Platfoot Strain' [6] are suggested to be involved in breeding programs to identify further gene markers for yellow fruit coloration. Heirlooms ' Aunt Ruby's German Green' [1] -due to its fruit taste and three red-fruited heirlooms of 'Black from Tula' [2] 'Cherokee Purple' [3] and 'German Johnson' [4] due to their high lycopene and β-carotene contents, seems to have high potential for more intensive recultivation purposes. The molecular profiling applied in this study also provides further use for metabolomics.