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Differential accumulation of flavonoids by tomato (Solanum lycopersicum) fruits tissues during maturation and ripening

Objective: Little is known about physiological functions of flavonoids, specifically in the course of maturation and ripening of fruits. Spatiotemporal changes in the levels of flavonoids were investigated in the present study with focus on possible functional differentiation of individual compounds as related to the maturation or ripening of tomato fruits. Methodology and results: The contents of flavonoids in different tissues of tomato fruits at increasing maturation and ripening stages were determined using high performance liquid chromatography – mass spectrometry. The levels of eriodictyol, kaempferol-glc-rhamnose, naringenin, naringenin-chalcone-hexose and quercetin-glc-rhamnose remained almost constant in the mesocarp and endocarp. The contents of eriodictyol, dicaffeoylquinic acid, naringenin and naringenin-chalcone-hexose significantly (P<0.05) increased in the epicarp from ripening stage 1 onwards. The concentration of dicaffeoyquinic acid increased significantly (P<0.05) in both the mesocarp and endocarp of tomato fruits from ripening stage 1 onwards. Gradual increases in the levels of caffeic-acid-hexose and caffeoylquinic acid in the epicarp and endocarp of tomatoes were observed. The level of kaempferol-glc-rhamnose decreased gradually in the epicarp. The content of quercetin-glc-rhamnose was always higher in the epicarp than in the mesocarp and endocarp. The results obtained indicated increases in the endogenous levels of some flavonoids in the epicarp (especially naringenin) with the onset of the ripening. There was also a gradual decrease and increase in the levels of respectively kaempferol-glc-rhamnose in the epicarp and caffeic-acid-hexose in the endocarp. Thus, an increase in the level of naringenin in the epicarp could be considered as physiological index for the ripening whereas high levels of kaempferol-glc-rhamnose in the epicarp and caffeic-acidhexose in the endocarp could serve as characteristic traits for respectively immature and red-ripe state of tomato fruits. On the whole, the results point to specific roles of individual flavonoids as some might be involved in the regulation of either the maturation or ripening of tomato fruits whereas others might functionally be needed throughout both processes, and that there would be a specialization of tissues in the synthesis of specific types of flavonoids. Conclusions and application findings: The degree of accumulation of flavonoids in tomato varied according to the nature of the tissue, and the maturation and ripening stages. It is hypothesized that an increase in Journal of Applied Biosciences 84:7674– 7681


MATERIALS AND METHODS
Plant material: Tomato (Solanum lycopersicum L. cv Balkonstar) plants were grown in a growth chamber at the Centre for Life and Food Sciences Weihenstephan of the Technical University of Munich (Germany) under a 16h/8h-photopperiod with a light intensity of 70±10 µmol/m 2 /s and constant temperature of 23±1 °C. Tomato fruits were harvested at different maturation stages ( Fig. 1), their pericarp, mesocarp and endocarp separated and stored at -20 °C for the study of spatiotemporal variations of the endogenous level of flavonoids. The tomato fruit tissues were then ground in a mortar under liquid nitrogen. One (1) g each was homogenized with a mixture containing 1 ml of methanol and 20 µl of 4-methylumbellyferyl-β-Dglucuronide dihydrate solution (9 mg/ml methanol) as internal standard. The mixture was centrifuged (5300 g, 30 min), 2 ml of supernatant then removed and concentrated by speed vacuum. The residue was resuspended in 150 µl methanol and centrifuged again for 10 min at 13400 g. The supernatant was removed, stored at 4 °C and subsequently analyzed high performance liquid chromatography -mass spectrometry. M3, ø = 3.0 cm; M4/R1, mature green (ø = 4.5 cm); R2, turning; R3, pinkish; R4, red-ripe; eq, equivalent; MUG, 4methylumbellyferyl-β-D-glucuronide dihydrate.
High performance liquid chromatography -mass spectrometry (LC-MS): An Agilent 1100 HPLC (Agilent Technologies, Waldbronn, Germany) equipped with a quaternary pump and a variable wavelength detector, and connected to a Bruker Daltonics Esquire 3000 Plus ion trap mass spectrometer (Bruker Daltonics, Bremen, Germany) was utilized. A C18 column (150 mm x 2.0 m, particle size 5 µm) held at 25 °C was used.
The mobile phase was a mixture of water (A) and methanol (B) both containing 0.1% formic acid. The flow rate was 0.2 ml min -1 . A gradient of 100% A for 5 min, 100% A to 60% A within 20 min, 60% A to 0% A in 5 min, 100% B for 7 min, 100% B to 0% B within 3 min and100% A for 10 min was applied. The detection wavelength was 280 nm. The electron spray ionization voltage of the mass spectrometer ranged from -4000 V to -500 V. Nitrogen was used as dry gas at temperature of 330 °C and a flow rate of 10.01 min -1 . The full scan mass spectra were measured in the range from m/z 50 to 800 with a scan resolution of 13000 m/z s -1 . The collision gas for the mass spectrometry was helium with a collision voltage of 1 V. Mass spectra were acquired in both positive and negative ionization modes. Analyses of LC-MS data were performed using the Bruker Daltonics software.  Statistical analyses: Group comparisons were made using One -Way Analysis of Variance (ANOVA) to determine if the variations among means were significantly greater than expected by chance. The Student -Newman -Keuls Test was used to compare means differences. In some few cases where differences between standard deviations of means were significant, the GraphPad Instat -software automatically proposed the use of rather the Nonparametric ANOVA and Kruskal-Wallis (as posttest).

RESULTS
Flavonoids and phenylpropanoids were analyzed in different tissues of tomato fruit at various maturation and ripening stages by LC-MS. Levels were calculated as mg-equivalents relative to the internal standard 4-methylumbellyferyl-β-D-glucuronide dihydrate. The endogenous level of caffeic-acid-hexose increased in all the investigated tissues during the growth and development of tomato fruits. From the maturation stage 3 onwards, the content of caffeic-acid-hexose was significantly higher in the endocarp than in the epicarp and mesocarp. At the ripening stage 4, the quantity of this compound found in the epicarp was significantly greater than in the mesocarp (Fig. 2). The concentration of caffeoylquinic acid slightly increased in all the three tissues of tomato fruits during the experimental period. From ripening stage 2 to ripening stage 4, the level of this compound was significantly higher in the epicarp than in the mesocarp (Fig. 3). The endogenous level of dicaffeoylquinic acid did not change in all the three tissues analyzed from the maturation stage 1 to the maturation stage 4. Thereafter, there were significant increases in the level of this metabolite in all the three tissues investigated. At the ripening stage 4, the endogenous concentration of dicaffeoylquinic acid was significantly higher in the epicarp and endocarp than in the mesocarp (Fig. 4). The eriodictyol content in the mesocarp and endocarp remained unchanged throughout the maturation and ripening of tomato fruits.
However, there was a significant increase in the concentration of this compound in the epicarp from ripening stage 3 to the ripening stage 4 (Fig. 5). A gradual decrease was observed in the level of kaempferol-glc-rhamnose in the epicarp during the growth and development of tomato fruits. No remarkable change could be observed in the concentration of this metabolite in the mesocarp and endocarp during the experimental period (Fig. 6). The endogenous level of naringerin remained nearly constant at low concentration in the epicarp from maturation stage 1 to stage 4, and then increased significantly from the latter stage onwards. Thus, the naringenin content in the epicarp at the ripening 4 was about ninetyfold higher than at the ripening stage 1. No increase in the concentration of naringenin in the mesocarp and endocarp could be observed during the growth and development of tomatoes (Fig. 7). The content of narigenin-chalcone-hexose remained nearly constant in the mesocarp and endocarp during the maturation and ripening of tomato fruits. However, this metabolite accumulated in the epicarp of tomato fruits increasingly from the maturation stage 4 onwards (Fig.  8). The level of quercetin-glc-rhamnose in all the three tissues studied remained almost unchanged throughout the experimental period. However, during the same period the quantity of quercetin-glc-rhamnose found in the epicarp was significantly higher than that found in other tomato fruit tissues analyzed (Fig. 9).

DISCUSSION
The levels of caffeic-acid-hexose, eriodictyol, dicaffeoylquinic acid, naringenin and naringeninchalcone-hexose in the epicarp of tomato fruits increased from ripening stage 1 onwards and this trend was more pronounced in the variation of naringenin content. On the contrary the level of kaempferol-glcrhamnose decreased continuously during the maturation (or growth and development) and ripening phases of tomatoes. Like in other fruits, the peel (epicarp) of tomato fruits is a tissue that undergoes one of the most perceptible changes (e.g. colour change) during transition from development to ripening (Gillapsy et al., 1993;Carrari and Fernie, 2006;Moco et al., 2007). Obviously, high levels of kaempferol-glcrhamnose are characteristic for green chloroplastcontaining tissues whereas high levels of naringenin, naringenin-chalcone-hexose, eriodictyol, caffeic-acidhexose and dicaffeoylquinic acid rather characterize coloured chromoplast-containing tomato fruit epicarps although these metabolite do not contribute to the colour. Kaempferol-glc-rhamnose may thus compete with other flavonoids, especially naringenin, for potential substrates. The physiological roles of kaempferol-glc-rhamnose may be confined to the early stage of growth and development of tomato fruits and those of naringenin and other flavonoids restricted to the ripening stages. In this context, kaempferol-glc-rhamnose may stimulate the biosynthesis of growth promoting hormones such as auxins and gibberellins that are massively produced at the cell division and expansion phases of tomato fruit development (Gillapsy et al., 1993;Osorio et al., 2013). The pathway of the biosynthesis of naringenin, naringenin-chalcone-hexose, eriodictyol, caffeic-acidhexose and dicaffeoylquinic acid may be diverted towards kaempferol-glc-rhamnose in the epicarp of tomato fruits during the development phase. Furthermore, since naringenin has been shown to competitively antagonize gibberellins (Phillips, 1962), its level and that of other flavonoids which function as growth inhibitors should be maintained at minimum values during the maturation of tomato fruits. Moreover, it is well known that the endogenous ethylene concentration peaks at the transition from breaker (mature green) to turning ripening stages of tomato fruits (Gillapsy et al., 1993;Carrrari and Fernie, 2006), a period where the levels of naringenin and some other flavonoids started to increase. Thus, these specific flavonoids may play important roles in the regulation of climacteric ethylene biosynthesis in tomato fruits. In return, ethylene may stimulate their biosynthesis in the epicarp of tomato fruits during the ripening stages. In fact, it is well established that ethylene, abscissic acid, jasmonic acid and methyljasmonate enhance the biosynthesis of flavonoids (Pirie and Mullins, 1976;Berhow, 1998;Buer et al., 2006;He et al., 2010). Those flavonoids whose levels increased gradually in the epicarp from ripening stage 1 onwards may be implicated in the prevention of the carotenoid photobleaching (Takanama, 1982). Naringenin could also be implicated in physiological mechanisms of the senescence prevention. In fact, Zhang et al. (2013) reported that enrichment of the anthocyanins (flavonoids) in tomato fruits led to a significant extension of their shelf-life by delaying over ripening and reducing susceptibility to Botrytis cinerea. However, in spite of the above discussed physiological and defensive roles of kaempferol-glc-rhamnose, naringenin, naringenin-chalcone-hexose, eriodictyol, caffeic-acid-hexose and dicaffeoylquinic acid during the development and ripening of tomato fruits, the exact mechanisms of their involvement in the control and regulation of some cardinal events that occur in the epicarp such as the greening and degreening are not yet exactly known. Apart from dicaffeoylquinic acid, there was no significant change in the levels of all flavonoids analyzed in the mesocarp during the experimental period. The levels of naringenin, naringenin-chalcone-hexose, eriodictyol, quercetin-glcrhamnose and kaempferol-glc-rhamnose in the endocarp remained nearly constant during the development and ripening of tomato fruits. However, significant increases in the contents of caffeic-acidhexose, caffeoylquinic acid and dicaffeoylquinic acid were observed in the endocarp. Contrarily to the mesocarp that seemingly does not undergo important morphological and physiological modifications, noticeable morphological and biochemical changes occurred in the endocarp as spatially delimited in the present study (Moco et al., 2007). This could explain why no remarkable changes in the levels of flavonoids could be observed in the mesocarp. Interestingly, caffeic acid derivatives were the only flavonoids whose contents significantly increased in the endocarp during the ripening of tomato fruits. This was a sign that the caffeic acid derivatives concerned may play special roles in the control and regulation of metabolic processes occurring in the endocarp of tomato fruits during the ripening stages. Like anthocyanins, caffeicacid-hexose may be involved in the mechanisms that control the senescence of tomato fruits . However, the precise physiological roles of caffeic acid derivatives in the endocarp of tomato fruits need still to be explained.

CONCLUSION
There were spatial and temporal variations in the accumulation of flavonoids in tomato fruits. Caffeicacid-hexose, kaempferol-glc-rhamnose and naringenin were the most concerned by these variations. Caffeic-acid-hexose and naringenin may play important physiological roles in the retardation of the over ripening.