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Serotonin content in fresh and processed tomatoes and its accumulation during fruit development
Scientia Horticulturae 214 (2017) 107–113
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Scientia Horticulturae journal homepage: www.elsevier.com/locate/scihorti
Serotonin content in fresh and processed tomatoes and its accumulation during fruit development Shohei Hano a,1 , Tomoki Shibuya a,1 , Nozomi Imoto a , Ayaka Ito a , Shunsuke Imanishi b , Hisashi Aso a , Yoshinori Kanayama a,∗ a b
Graduate School of Agricultural Science, Tohoku University, 468-1 Aramaki Aza-Aoba, Aoba-ku, Sendai 980-0845, Japan NARO Institute of Vegetable and Floriculture Science, Ano, Tsu 514-2392, Japan
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Article history: Received 22 August 2016 Received in revised form 9 November 2016 Accepted 11 November 2016 Keywords: Fruit Serotonin Solanum lycopersicum Tomato Tryptophan decarboxylase
a b s t r a c t Serotonin is an aromatic amine neurotransmitter in the central nervous system; however, approximately 98% of serotonin is synthesized and stored in the peripheral system. We analyzed tomatoes (Solanum lycopersicum), which are relatively rich in serotonin, because serotonin has been found to have antiobesity effects in the peripheral system. Serotonin content was very low in processed tomato products, whereas fresh tomatoes were much richer in serotonin. Serotonin content increased in all fruit tissues during tomato fruit development, reaching maximum levels at the ripe stage. Differences in serotonin content were relatively small among fruit tissues at the ripe stage. During storage, serotonin content did not decrease at either room temperature or at the lower temperature (4◦ C). Sequence and expression analyses were performed for tryptophan decarboxylase (TDC) and tryptamine 5-hydroxylase (T5H) genes, which could be related to the serotonin biosynthesis pathway from tryptophan. As a result, expression of SlTDC1, one of the tomato putative TDC family genes, and SlT5H, the tomato putative T5H homolog, likely corresponds to an increase in serotonin content during fruit development. The results suggest that fresh tomatoes are a promising source of serotonin, and SlTDC1 and SlT5H might be involved in physiological mechanisms of serotonin accumulation. © 2016 Elsevier B.V. All rights reserved.
1. Introduction Serotonin is an aromatic amine neurotransmitter that controls several physiological functions, such as mood, sleep, and anxiety in animals and humans (Veenstra-VanderWeele et al., 2000). However, approximately 98% of serotonin is synthesized and stored in the peripheral system. Although serotonin in the peripheral system also has some functions, the functions are not yet fully understood (Watanabe et al., 2011). Recently, it has been found that in mice, serotonin enhances lipid metabolism by stimulating the excretion of bile in the peripheral system (Watanabe et al., 2010, 2011, 2016). Serotonin intake via food cannot cross the blood-brain barrier, and thus acts in the peripheral system. Xiao et al. (1998) reported that
Abbreviations: DAF, days after flowering; PLP, pyridoxal phosphate; SSC, soluble solids content; TDC, tryptophan decarboxylase; TYDC, tyrosine decarboxylase; T5H, tryptamine 5-shydroxylase. ∗ Corresponding author at: Graduate School of Agricultural Science, Tohoku University, 468-1 Aramaki Aza-Aoba, Aoba-ku, Sendai 980-0845, Japan. E-mail address: [email protected] (Y. Kanayama). 1 These authors equally contributed to the paper. http://dx.doi.org/10.1016/j.scienta.2016.11.009 0304-4238/© 2016 Elsevier B.V. All rights reserved.
intake of banana, which is relatively rich in serotonin, increases the blood serotonin level. Therefore, considering the anti-obesity effects of serotonin, it is important in horticultural science to focus on vegetables and fruits as sources of serotonin and to investigate serotonin content, synthesis, and accumulation. Serotonin content in tomato (Solanum lycopersicum) appears to be relatively high among vegetables (Feldman and Lee, 1985). In addition, tomatoes are rich in lycopene, which has an antioxidant activity (Hu et al., 2013), and 13-oxo-9,11-octadecadienoic acid in tomato juice regulates lipid metabolism (Kim et al., 2012). Tomatoes are the most abundantly produced fruit globally (FAOSTAT, http://faostat.fao.org). Therefore, in a health-oriented modern society, providing new information regarding a functional ingredient, such as serotonin, in tomatoes may result in increasing the value of tomatoes due to health benefits, thereby further increasing tomato consumption. In animals, serotonin is synthesized by tryptophan hydroxylase, which catalyzes the hydroxylation of tryptophan to produce 5-hydroxytryptophan, followed by aromatic amino acid decarboxylase to form serotonin, with tryptophan hydroxylase acting as a rate limiting enzyme (Veenstra-VanderWeele et al., 2000). In plants (such as rice and pepper), it is reported that serotonin is synthesized by tryptophan decarboxylase (TDC), which catalyzes
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Fig. 3. Serotonin content in various fruit tissues. Exocarp, mesocarp, seed and jelly, placenta, and columella tissues were sampled at ten days after flowering (DAF), 20 DAF, 30 DAF, and the ripe stage, and analyzed. Each value was determined in three independent biological replicates and indicate a mean ± standard error (n = 3). Different lowercase letters indicate significant differences among fruit developmental stages in each tissue and different uppercase letters indicate significant differences among tissues at the ripe stage at P < 0.05 according to Tukey’s test.
Fig. 4. Effect of salt stress on soluble solids and serotonin content in fruit. Plants were supplied with 80 mM or 160 mM NaCl and fruit was harvested at the ripe stage. Pericarp tissues were used for the analysis and each value was determined in three independent biological replicates. Values indicate means ± standard error (n = 3). Values with the same letter were not significantly different at P < 0.05 according to Tukey’s test. The SSC data in control and 80 mM is the same as that in Ikeda et al. (2016).
Fig. 1. Serotonin content in fresh tomatoes. The nine cultivars or brands of tomatoes purchased and used for the analysis were cherry tomatoes (S1, S2, S3, S4), middle-sized tomatoes (M1, M2), and large-sized tomatoes (L1, L2 L3). SSC was measured for reference (A), as well as serotonin content (B). SSC for reference (C) and serotonin content (D) in fresh fruit were also measured for five and ten days at 4 ◦ C or 25 ◦ C, after the harvest at breaker stage. Pericarp tissues were used for the analysis and each value was determined in three independent biological replicates. Values indicate means ± standard error (n = 3). Values with the same letter were not significantly different at P < 0.05 according to Tukey’s test.
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the decarboxylation of tryptophan to form tryptamine, following which catalysis by tryptamine 5-hydroxylase (T5H) forms serotonin (Kang et al., 2009a; Park et al., 2009). In rice leaves, senescence increases TDC gene expression and serotonin content, and the overexpression of the TDC gene increases serotonin content and delays senescence (Kang et al., 2009a). Tryptamine synthesized by TDC is converted to serotonin by T5H, a cytochrome P450 enzyme (Fujiwara et al., 2010). Upon pathogen infection in pepper fruit, the expression of the TDC gene and serotonin content are increased to induce responses to infection (Park et al., 2009). Despite these previous findings, very few studies have focused on serotonin accumulation and the expression of serotonin synthesis-related genes during fruit development. In this study, to evaluate tomato fruit as a promising source of serotonin, we measured serotonin content in both fresh and processed tomatoes, and compared serotonin content in various fruit tissues. We also investigated serotonin synthesis-related genes to understand the determining factors of serotonin accumulation during fruit development.
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Fig. 2. Serotonin content in processed tomatoes. Tomato ketchup (K1, K2, K3), tomato juice (J1, J2), and canned whole tomatoes (W1, W2) were used for the analysis, and two or three items in each type of processed tomato were from different companies. Each value was determined in three independent extractions. Values indicate means ± standard error (n = 3). Values with the same letter were not significantly different at P < 0.05 according to Tukey’s test.
2. Materials and methods 2.1. Determination of serotonin content in fresh and processed tomatoes Fresh fruits of cherry tomatoes, middle-, and large-sized tomatoes were purchased from local markets, and their pericarp tissues
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Fig. 5. Alignment of amino acid sequences of SlTDC1, SlTDC2, and PepTDC1 (A) and the phylogenetic tree, based on the amino acid sequences of tryptophan decarboxylase (TDC) and tyrosine decarboxylase (TYDC) homologs (B). The alignment was constructed by Genetyx and ClustalW2 programs. Identical amino acids are denoted with black or gray. The conserved regions found in pyridoxal phosphate (PLP)-dependent decarboxylases are underlined. The arrow indicates a putative PLP-binding site (K). The tree was constructed by the neighbor-joining method, using the ClustalW2 program. Branch numbers refer to the percentage of replicates that support the branch, using the bootstrap method (1000 replicates). Species and accession numbers were as follows: OsTDC1 (Oryza sativa, AK069031), OsTDC3 (O. sativa, NM 001067504), SlTDC1 (S. lycopersicum, AK327027), SlTDC2 (S. lycopersicum, XM 004243286), PepTDC1
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Tomato ‘Ailsa Craig’ was grown in pots in a greenhouse according to the method of Ikeda et al. (2013), and exocarp, mesocarp, seed and jelly, placenta, and columella tissues were sampled at various stages of fruit development. For salt stress treatment, plants were supplied with 80 or 160 mM NaCl every week after flowering, as described previously (Yin et al., 2010). Pericarp tissues were sampled at the ripe stage. Soluble solids and serotonin content were measured as described in 2.1.
in large-sized tomatoes, although some of the differences were not statistically significant. In contrast, there was no significant difference in serotonin content between various sized tomatoes, except tomato M2 (Fig. 1B). Tomato M2 grown under abiotic stress conditions to increase SSC showed high serotonin content as well as high SSC. Once the tomatoes were harvested, the stability of serotonin was investigated for both 4◦ C and 25◦ C for a period of 10 days. As a result, serotonin content did not decrease during the period at either temperature, with SSC measured for reference (Fig. 1C, D). The stability of serotonin during storage for a similar period is supported by the previous study using banana (Adao and Gloria, 2005). Fruit ripening presumably progressed during the period because SSC increased. The following processed tomato products were used for evaluation of serotonin content (Fig. 2): 1) tomato ketchup (three brands); 2) tomato juice (two brands), and; 3) canned whole tomatoes (two brands). The serotonin content in juice was higher than that in ketchup and canned whole tomatoes; however, the average serotonin content in the processed tomato products was 0.06 mg g−1 FW, which was approximately a hundred times lower than that found in fresh tomatoes.
2.3. Sequence analysis of the TDC and T5H homologs in tomato
3.2. Serotonin accumulation during fruit development
Tomato sequences homologous to the pepper TDC gene (PepTDC1, Park et al., 2009) and the rice T5H gene (Fujiwara et al., 2010) were sought using the National Center for Biotechnology Information (NCBI) Basic Local Alignment Search Tool (BLAST) (http://www.ncbi.nlm.nih.gov/), and the three complementary DNA (cDNA) sequences found were named SlTDC1, SlTDC2, and SlT5H. Their sequences in the tomato genome were confirmed using the Sol Genomics Network (http://solgenomics.net/ ) database. Sequence alignment and phylogenetic trees were prepared using Genetyx software and Multiple Sequence Alignment by ClustalW2. A protein domain search was performed using InterProScan (http://www.ebi.ac.uk/interpro/).
An increase in serotonin content was observed during fruit development in all fruit tissues (Fig. 3). Serotonin content was lowest at 10 days after flowering (DAF) and was highest at the ripe stage. Serotonin content was very low in seed and jelly, placenta, and columella tissues at 10 and 20 DAF; yet, serotonin content increased remarkedly at 30 DAF. Serotonin content in mesocarp tissues was highest in various fruit tissues at the ripe stage, although differences in serotonin content were not large between tissues. Serotonin content in pericarp at the ripe stage increased under the stress condition of 160 mM NaCl when SSC was increased (Fig. 4).
were used for the analysis. Processed tomatoes, such as ketchup, juice, and canned whole tomatoes were also purchased from local markets. To evaluate changes in serotonin content during storage, ‘M82’ tomato plants were grown in a planting bed in a greenhouse according to the method of Ikeda et al. (2013), and breaker stage fruit were stored at 4◦ C and 25◦ C. Serotonin content was measured by the Serotonin EIA Kit (Beckman Coulter) according to the manufacturer’s instructions. Soluble solids content (SSC; Brix) in fresh tomatoes was measured for reference, using a Brix meter. 2.2. Determination of serotonin content during tomato fruit development
3.3. Sequence analysis of cDNA encoding SlTDC1, SlTDC2, and SlT5H
2.4. Expression analysis of SlTDC1, SlTDC2, and SlT5H Total RNA was extracted from fruit pericarp tissues at each stage, and removal of genomic DNA, reverse transcription, and real-time PCR were performed according to the methods of Mohammed et al. (2012). The following gene-specific primer sets were designed using the Primer 3 software: 5 -TAGTTCCGCAGCGTTCATTG-3 5 -CGAGTTTACGATCACGTGCAG-3 for SlTDC1; 5 and CGTTAAGTCCGATCCCATGT-3 and 5 -CTCAGTGAACGTCGCACCTA3 for SlTDC2; 5 -GTTGTCCAGGTTATGCTCTTGG-3 and 5 -CCTTCCCATGGCTACAATCAAC-3 for SlT5H; 5 and 5 -ACGTCCCTGACAATTTCACTGTATGCCAGTGGTCGT-3 CTCG-3 for SlActin (Accession No. BT012695). 3. Results 3.1. Serotonin contents of fresh and processed tomatoes Fresh tomatoes of various fruit sizes were used for the evaluation of serotonin content (Fig. 1). SSC, which is one of the most important qualities of tomato, was also determined for reference. SSC in cherry tomato S1 and S2 and middle-sized tomato M2 were significantly higher than that in large-sized tomatoes (Fig. 1A). SSCs in other cherry and middle-sized tomatoes were also higher than
Tomato sequences homologous to one of the pepper TDC genes (PepTDC1, Park et al., 2009) were sought using NCBI BLAST, resulting in two cDNA sequences being identified, which were named SlTDC1 and SlTDC2. It was also confirmed in the Sol Genomics Network database that two putative TDC genes exist in the tomato genome. The amino acid sequences of SlTDC1 and SlTDC2 were 85.7% and 83.3% identical to that of PepTDC1, respectively, whereas the SlTDC1 sequence was 89.3% identical to that of SlTDC2 (Fig. 5A). Using InterProScan, SlTDC1, and SlTDC2 were found to belong to aromatic amino acid decarboxylases, which are pyridoxal phosphate (PLP)-dependent decarboxylases. The conserved regions of PLP-dependent decarboxylases and PLP-binding sites were conserved in SlTDC1 and SlTDC2, as well as in PepTDC1 (Park et al., 2009). Tomato sequences homologous to another pepper TDC gene (PepTDC2) were also sought; however, no sequences showing good homology were found. A phylogenetic tree was constructed based on the amino acid sequences of TDC and tyrosine decarboxylase (TYDC), which belong to aromatic amino acid decarboxylases along with TDC (Fig. 5B). In the tree, SlTDC1, SlTDC2, and PepTDC1 fell into the TDC group, whereas PepTDC2 and OsTDC2 fell into the TYDC group. Tomato sequences homologous to the rice T5H gene (OsT5H, Fujiwara et al., 2010) were sought using NCBI BLAST, and a cDNA
(Capsicum annuum, FJ710789), PepTDC2 (C. annuum, FJ710788), CrTDC1 (Catharanthus roseus, M25151), OpTDC1 (Ophiorrhiza pumila, AB086168), CaTDC1 (Camptotheca acuminata, U73656), CaTDC2 (C. acuminata, U73657), AtTYDC (Arabidopsis thaliana, AY074539), OsTYDC (O. sativa, AK065830), OsTDC2 (O. sativa, AK103253), PsTYDC1 (Papaver somniferum, U08597), PsTYDC2 (P. somniferum, U08598), PcTYDC2 (Petroselinum crispum, M96070), and PcTYDC4 (P. crispum, M95685).
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Fig. 6. Alignment of amino acid sequences of SlT5H and OsT5H (A) and the phylogenetic tree, based on the amino acid sequences of SlT5H, OsT5H and tomato and rice proteins that belong to the cytochrome P450 groups close to the CYP71 group (B). The alignment was constructed by Genetyx and ClustalW2 programs. Identical amino acids are denoted with black. The K-helix motif and heme-binding motif are marked by dotted and thick underlines, respectively. The arrow indicates the glycine residue in the heme-binding motif, which is essential for T5H functioning (Fujiwara et al., 2010). The tree was constructed by the neighbor-joining method, using the ClustalW2 program. Branch numbers refer to the percentage of replicates that support the branch using the bootstrap method (1000 replicates). Species and accession numbers were as follows: OsT5H (O. sativa, AK071599), SlT5H (S. lycopersicum, NM 001247918). Other accession codes are from the cytochrome P450 homepage (http://drnelson.uthsc.edu/CytochromeP450.html).
sequence was identified, named SlT5H. It was confirmed in the Sol Genomics Network database that one putative T5H gene exists in the tomato genome. The amino acid sequence of SlT5H was 51.4% identical to that of OsT5H (Fig. 6A). According to Fujiwara et al. (2010) and the Sol Genomics Network database, OsT5H and SlT5H both belong to the CYP71 group of cytochrome P450. The K-helix motif and heme-binding motif were conserved in both SlT5H and OsT5H. The glycine residue in the heme-binding motif, which is essential for T5H function (Fujiwara et al., 2010), was also conserved in both SlT5H and OsT5H. A phylogenetic tree was constructed based on the amino acid sequences of SlT5H, OsT5H, and tomato and rice proteins belonging to the cytochrome P450 groups
close to the CYP71 group (Fig. 6B). Phylogenetically, SlT5H was most similar to OsT5H.
3.4. Comparison between changes in serotonin content and the SlTDC1, SlTDC2, and SlT5H mRNA levels during tomato fruit development The SlTDC1, SlTDC2, and SlT5H levels were measured in pericarp tissues during fruit development and compared with serotonin content (Fig. 7). Serotonin content increased until breaker stage and did not change thereafter. The SlTDC1 mRNA level increased until 35 DAF and then decreased. The SlTDC2 mRNA level was constantly
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Fig. 7. Changes in serotonin content (A) and the SlTDC1 (B), SlTDC2 (C), and SlT5H (D) mRNA levels during tomato fruit development. Serotonin content data (A) in 10, 20, 30 DAF, and Ripe is the same as that in Fig. 3 (mesocarp). Pericarp tissues were used for the analysis and each value was determined in three independent biological replicates. Data (B, C, D) shows the relative expression levels, normalized against SlActin. Values indicate means ± standard error (n = 3). Values with the same letter were not significantly different in each plot at P < 0.05 according to Tukey’s test.
very low, and was 4400 times lower than the SlTDC1 mRNA level at 35 DAF. The SlT5H mRNA remained at similar levels until the breaker stage, after which it decreased to the lowest level. 4. Discussion Serotonin contents were measured in fresh and processed tomatoes for comparison because knowledge of this comparison remains incomplete. Serotonin content was found to be very low in processed tomato products including juice, ketchup, and canned whole tomatoes. The results suggest that processing such as heat treatment might decrease serotonin content in tomatoes. Therefore, fresh tomatoes were a far better source of serotonin. The average serotonin content was 6.4 g g−1 FW in fresh tomatoes of various fruit sizes. This value of serotonin content was higher than that
of previous tomato recordings (3.2 g g−1 FW), and third-highest recorded in fresh fruits and vegetables (Feldman and Lee, 1985). It was also much higher than that recorded in other popular vegetables such as broccoli (0.2 g g−1 FW) and eggplant (0.2 g g−1 FW) (Feldman and Lee, 1985). Studies on serotonin accumulation in various fruit tissues throughout fruit development remain poorly represented in the literature. Serotonin content increased in all fruit tissues during fruit development and reached maximum levels at the ripe stage, when they are also usually the most palatable. Differences in serotonin content were relatively small among fruit tissues at the ripe stage, and serotonin content in mesocarp, which is the main fruit tissue, was highest among all fruit tissues. With regards to other functional food ingredients, differences among tomato fruit tissues in ascorbic acid content have been noted to be within a factor of two, whereas lycopene content has varied by a factor greater than ten (Moco et al., 2007). Our results and those of the previous study suggest that tissue localization of functional food ingredients varies. During storage, serotonin content was not reduced at either room temperature or the low temperature. Many beneficial components of tomato fruit have been shown to decrease during ripening and storage (Oms-Oliu et al., 2011), even though sugar content increases fractionally, as it also did in our study. These results suggest that fresh tomatoes are a promising source of serotonin. Generally, SSC corresponded to fruit size, except in one tomato type (M2) grown under abiotic stress to increase SSC. Serotonin content did not correspond to fruit size. Serotonin content in tomato fruit was also increased in our cultivation under salt stress conditions. Cultivation under abiotic stress conditions appears to be a promising method for producing high quality fruit, because in addition to serotonin production in the present study, the method may promote metabolism of sugar and other useful components such as amino acids (Ikeda et al., 2016). Tomato putative TDC genes, SlTDC1 and SlTDC2, were identified based on the amino acid sequence of PepTDC1 because PepTDC1 has been proven to be a TDC using the recombinant protein (Park et al., 2009). Sequence analysis confirmed that SlTDC1 and SlTDC2 belong to the TDC gene group. Two aromatic amino acid decarboxylase genes in pepper, PepTDC1 and PepTDC2 (Park et al., 2009), were classified into the TDC and TYDC gene groups, respectively, whereas both genes in tomato, SlTDC1 and SlTDC2, were classified into the TDC gene group. Tomato putative T5H gene, SlT5H, was also identified based on the amino acid sequence of OsT5H, because OsT5H has been proven to be a T5H, using the recombinant protein (Fujiwara et al., 2010). Sequence analysis confirmed that SlT5H belongs to the T5H gene group, i.e., the CYP71 group of cytochrome P450. Collectively, three genes that could be related to serotonin synthesis in tomato fruit were identified, and their expression was analyzed with serotonin accumulation. The SlTDC1 mRNA level increased with serotonin content during tomato fruit development, whereas the SlTDC2 mRNA level was constantly very low. SlTDC2 mRNA was also undetectable in the transcriptome analysis using custom DNA microarrays designed for the comprehensive array, consisting of probe sets based on the whole-genome sequence of tomato (Ikeda et al., 2016). The SlTDC2 mRNA was higher in leaves than in fruit (data not shown), suggesting its primary role in leaves rather than in fruit. A decrease in the SlTDC1 mRNA level after 35 DAF likely corresponds to the fact that serotonin content did not increase during the same period. Collectively, SlTDC1 might play a role in serotonin accumulation in tomato fruit. The SlT5H mRNA level was maintained from 15 DAF to breaker stage in tomato fruit, suggesting that SlT5H might play a role in serotonin synthesis from tryptamine, which SlTDC1 synthesized from tryptophan. TDC could be a key enzyme in serotonin synthesis
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in rice leaves, whereas the T5H gene is constitutively expressed (Kang et al., 2009a). Our results might support the previous study, although SlT5H could be the determinant of serotonin content in two tomato genotypes (Ikeda et al., 2016). Some studies on the physiological roles of serotonin in plants, such as in rice and pepper, found serotonin to be related to biotic and abiotic stress (Ishihara et al., 2008; Kang et al., 2009a; Park et al., 2009; Fujiwara et al., 2010). Serotonin increases from the normal level through stimulation by stress, and in these cases, to serve functions. Serotonin content in ripe tomato fruit in the present study was obviously higher than that recorded at the normal level in previous studies, and serotonin has high radical scavenging activity (Kang et al., 2009a). Therefore, the role of accumulated serotonin in tomato fruit is interestingly varied, and further studies will be necessary to clarify how its benefits can be optimized. In addition to serotonin, its metabolites such as melatonin and phenylpropanoid amides have also been reported to have physiological roles in biotic and abiotic stress responses in tomato (Kang et al., 2009b; Okazaki et al., 2009). Therefore, the accumulation of serotonin metabolites as well as serotonin should be investigated. The improvement of quality as well as the enhancement of stress tolerance have been proposed in pioneer work regarding metabolic engineering of tomato secondary metabolites (Davidovich-Rikanati et al., 2007), and the same type of study can be performed using SlTDC1. To the best of our knowledge, the present study is the first detailed study on serotonin accumulation during fruit development. The results revealed that fresh tomatoes are much more beneficial for serotonin intake than processed tomatoes, and serotonin accumulates during fruit development. We also suggest that SlTDC1, which is one of the tomato putative TDC family genes, as well as SlT5H, might play a role in the accumulation of serotonin. Further studies including transgenic experiments will be necessary to provide direct evidence for the functions of these enzymes because their functions were discussed using only sequence homology and expression data in this study. It is predicted that serotonin intake through daily consumption of vegetables and fruits can have anti-obesity effects, as it has been shown that daily consumption of banana, which similar to tomato and is rich in serotonin, increases serotonin in the bloodstream (Xiao et al., 1998). Our results could also be useful for the evaluation of tomato fruit quality in terms of serotonin content and the development of cultivation and breeding methods for serotonin-rich tomatoes. Acknowledgements The authors thank Hiroki Ikeda and Motoki Sato for their help and the National BioResource Project tomato (NBRP tomato) for information. This work was supported by the Research Project on Development of Agricultural Products and Foods with Health-promoting benefits (NARO) and Grants-in-Aid for Scientific Research [24248006]. References Adao, R.C., Gloria, M.B.A., 2005. Bioactive amines and carbohydrate changes during ripening of ‘Prata’ banana (Musa acuminata×M. balbisiana). Food Chem. 90, 705–711. Davidovich-Rikanati, R., Sitrit, Y., Tadmor, Y., Iijima, Y., Bilenko, N., Bar, E., Carmona, B., Fallik, E., Dudai, N., Simon, J.E., Pichersky, E., Lewinsohn, E.,2007.
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Enrichment of tomato flavor by diversion of the early plastidial terpenoid pathway. Nat. Biotechnol. 25, 899–901. Feldman, J.M., Lee, E.M., 1985. Serotonin content of foods: effect on urinary excretion of 5-hydroxyindoleacetic acid. Am. J. Clin. Nutr. 42, 639–643. Fujiwara, T., Maisonneuve, S., Isshiki, M., Mizutani, M., Chen, L., Wong, H.L., Kawasaki, T., Shimamoto, K., 2010. Sekiguchi lesion gene encodes a cytochrome P450 monooxygenase that catalyzes conversion of tryptamine to serotonin in rice. J. Biol. Chem. 285, 11308–11313. Hu, W., Dai, D., Li, W., 2013. Anti-aging effect of Blakeslea trispora powder on adult mice. Biotechnol. Lett. 35, 1309–1315. Ikeda, H., Hiraga, M., Shirasawa, K., Nishiyama, M., Kanahama, K., Kanayama, Y., 2013. Analysis of a tomato introgression line, IL8-3, with increased Brix content. Sci. Hortic. 153, 103–108. Ikeda, H., Shibuya, T., Imanishi, S., Aso, H., Nishiyama, M., Kanayama, Y., 2016. Dynamic metabolic regulation by a chromosome segment from a wild relative during fruit development in a tomato introgression line, IL8-3. Plant Cell Physiol. 57, 1257–1270. Ishihara, A., Hashimoto, Y., Tanaka, C., Dubouzet, J.G., Nakao, T., Matsuda, F., Nishioka, T., Miyagawa, H., Wakasa, K., 2008. The tryptophan pathway is involved in the defense responses of rice against pathogenic infection via serotonin production. Plant J. 54, 481–495. Kang, K., Kim, Y.S., Park, S., Back, K., 2009a. Senescence-induced serotonin biosynthesis and its role in delaying senescence in rice leaves. Plant Physiol. 150, 1380–1393. Kang, K., Lee, K., Sohn, S.O., Park, S., Lee, S., Kim, S.Y., Kim, Y.S., Back, K., 2009b. Ectopic expression of serotonin N-hydroxycinnamoyltransferase and differential production of phenylpropanoid amides in transgenic tomato tissues. Sci. Hortic. 120, 504–510. Kim, Y., Hirai, S., Goto, T., Ohyane, C., Takahashi, H., Tsugane, T., Konishi, C., Fujii, T., Inai, S., Iijima, Y., Aoki, K., Shibata, D., Takahashi, N., Kawada, T., 2012. Potent PPAR␣ activator derived from tomato juice, 13-oxo-9, 11-octadecadienoic acid, decreases plasma and hepatic triglyceride in obese diabetic mice. PLoS One 7, e31317. Moco, S., Capanoglu, E., Tikunov, Y., Bino, R.J., Boyacioglu, D., Hall, R.D., Vervoort, J., De Vos, R.C.H., 2007. Tissue specialization at the metabolite level is perceived during the development of tomato fruit. J. Exp. Bot. 58, 4131–4146. Mohammed, S.A., Nishio, S., Takahashi, H., Shiratake, K., Ikeda, H., Kanahama, K., Kanayama, Y., 2012. Role of vacuolar H+ -inorganic pyrophosphatase in tomato fruit development. J. Exp. Bot. 63, 5613–5621. Okazaki, M., Higuchi, K., Hanawa, Y., Shiraiwa, Y., Ezura, H., 2009. Cloning and characterization of a Chlamydomonas reinhardtii cDNA arylalkylamine N-acetyltransferase and its use in the genetic engineering of melatonin content in the Micro-Tom tomato. J. Pineal Res. 46, 373–382. Oms-Oliu, G., Hertog, M.L.A.T.M., Van de Poel, B., Ampofo-Asiama, J., Geeraerd, A.H., Nicola¨i, M., 2011. Metabolic characterization of tomato fruit during preharvest development, ripening, and postharvest shelf-life. Postharvest Biol. Technol. 62, 7–16. Park, S., Kang, K., Lee, K., Choi, D., Kim, Y.S., Back, K., 2009. Induction of serotonin biosynthesis is uncoupled from the coordinated induction of tryptophan biosynthesis in pepper fruits (Capsicum annuum) upon pathogen infection. Planta 230, 1197–1206. Veenstra-VanderWeele, J., Anderson, G.M., Cook, E.H., 2000. Pharmacogenetics and the serotonin system: initial studies and future directions. Eur. J. Pharmacol. 410, 165–181. Watanabe, H., Akasaka, D., Sato, H., Ogasawara, H., Sato, K., Miyake, M., Saito, K., Takahashi, Y., Kanaya, T., Takakura, I., Hondo, T., Chao, G., Rose, M.T., Ohwada, S., Watanabe, K., Yamaguchi, T., Aso, H., 2010. Peripheral serotonin enhances lipid metabolism by accelerating bile acid turnover. Endocrinology 151, 4776–4786. Watanabe, H., Rose, M.T., Aso, H., 2011. Role of peripheral serotonin in glucose and lipid metabolism. Curr. Opin. Lipidol. 22, 186–191. Watanabe, H., Nakano, T., Saito, R., Akasaka, D., Saito, K., Ogasawara, H., Minashima, T., Miyazawa, K., Kanaya, T., Takakura, I., Inoue, N., Ikeda, I., Chen, X., Miyake, M., Kitazawa, H., Shirakawa, H., Sato, K., Tahara, K., Nagasawa, Y., Rose, M.T., Ohwada, S., Watanabe, K., Aso, H., 2016. Serotonin improves high fat diet induced obesity in mice. PLoS One 11, e0147143. Xiao, R., Beck, O., Hjemdahl, P., 1998. On the accurate measurement of serotonin in whole blood. Scand. J. Clin. Lab. Invest. 58, 505–510. Yin, Y.G., Kobayashi, Y., Sanuki, A., Kondo, S., Fukuda, N., Ezura, H., Sugaya, S., Matsukura, C., 2010. Salinity induces carbohydrate accumulation and sugarregulated starch biosynthetic genes in tomato (Solanum lycopersicum L. cv. ‘Micro-Tom’) fruits in an ABA and osmotic stress-independent manner. J. Exp. Bot 61, 563–574.
Contents lists available at ScienceDirect
Scientia Horticulturae journal homepage: www.elsevier.com/locate/scihorti
Serotonin content in fresh and processed tomatoes and its accumulation during fruit development Shohei Hano a,1 , Tomoki Shibuya a,1 , Nozomi Imoto a , Ayaka Ito a , Shunsuke Imanishi b , Hisashi Aso a , Yoshinori Kanayama a,∗ a b
Graduate School of Agricultural Science, Tohoku University, 468-1 Aramaki Aza-Aoba, Aoba-ku, Sendai 980-0845, Japan NARO Institute of Vegetable and Floriculture Science, Ano, Tsu 514-2392, Japan
a r t i c l e
i n f o
Article history: Received 22 August 2016 Received in revised form 9 November 2016 Accepted 11 November 2016 Keywords: Fruit Serotonin Solanum lycopersicum Tomato Tryptophan decarboxylase
a b s t r a c t Serotonin is an aromatic amine neurotransmitter in the central nervous system; however, approximately 98% of serotonin is synthesized and stored in the peripheral system. We analyzed tomatoes (Solanum lycopersicum), which are relatively rich in serotonin, because serotonin has been found to have antiobesity effects in the peripheral system. Serotonin content was very low in processed tomato products, whereas fresh tomatoes were much richer in serotonin. Serotonin content increased in all fruit tissues during tomato fruit development, reaching maximum levels at the ripe stage. Differences in serotonin content were relatively small among fruit tissues at the ripe stage. During storage, serotonin content did not decrease at either room temperature or at the lower temperature (4◦ C). Sequence and expression analyses were performed for tryptophan decarboxylase (TDC) and tryptamine 5-hydroxylase (T5H) genes, which could be related to the serotonin biosynthesis pathway from tryptophan. As a result, expression of SlTDC1, one of the tomato putative TDC family genes, and SlT5H, the tomato putative T5H homolog, likely corresponds to an increase in serotonin content during fruit development. The results suggest that fresh tomatoes are a promising source of serotonin, and SlTDC1 and SlT5H might be involved in physiological mechanisms of serotonin accumulation. © 2016 Elsevier B.V. All rights reserved.
1. Introduction Serotonin is an aromatic amine neurotransmitter that controls several physiological functions, such as mood, sleep, and anxiety in animals and humans (Veenstra-VanderWeele et al., 2000). However, approximately 98% of serotonin is synthesized and stored in the peripheral system. Although serotonin in the peripheral system also has some functions, the functions are not yet fully understood (Watanabe et al., 2011). Recently, it has been found that in mice, serotonin enhances lipid metabolism by stimulating the excretion of bile in the peripheral system (Watanabe et al., 2010, 2011, 2016). Serotonin intake via food cannot cross the blood-brain barrier, and thus acts in the peripheral system. Xiao et al. (1998) reported that
Abbreviations: DAF, days after flowering; PLP, pyridoxal phosphate; SSC, soluble solids content; TDC, tryptophan decarboxylase; TYDC, tyrosine decarboxylase; T5H, tryptamine 5-shydroxylase. ∗ Corresponding author at: Graduate School of Agricultural Science, Tohoku University, 468-1 Aramaki Aza-Aoba, Aoba-ku, Sendai 980-0845, Japan. E-mail address: [email protected] (Y. Kanayama). 1 These authors equally contributed to the paper. http://dx.doi.org/10.1016/j.scienta.2016.11.009 0304-4238/© 2016 Elsevier B.V. All rights reserved.
intake of banana, which is relatively rich in serotonin, increases the blood serotonin level. Therefore, considering the anti-obesity effects of serotonin, it is important in horticultural science to focus on vegetables and fruits as sources of serotonin and to investigate serotonin content, synthesis, and accumulation. Serotonin content in tomato (Solanum lycopersicum) appears to be relatively high among vegetables (Feldman and Lee, 1985). In addition, tomatoes are rich in lycopene, which has an antioxidant activity (Hu et al., 2013), and 13-oxo-9,11-octadecadienoic acid in tomato juice regulates lipid metabolism (Kim et al., 2012). Tomatoes are the most abundantly produced fruit globally (FAOSTAT, http://faostat.fao.org). Therefore, in a health-oriented modern society, providing new information regarding a functional ingredient, such as serotonin, in tomatoes may result in increasing the value of tomatoes due to health benefits, thereby further increasing tomato consumption. In animals, serotonin is synthesized by tryptophan hydroxylase, which catalyzes the hydroxylation of tryptophan to produce 5-hydroxytryptophan, followed by aromatic amino acid decarboxylase to form serotonin, with tryptophan hydroxylase acting as a rate limiting enzyme (Veenstra-VanderWeele et al., 2000). In plants (such as rice and pepper), it is reported that serotonin is synthesized by tryptophan decarboxylase (TDC), which catalyzes
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Fig. 3. Serotonin content in various fruit tissues. Exocarp, mesocarp, seed and jelly, placenta, and columella tissues were sampled at ten days after flowering (DAF), 20 DAF, 30 DAF, and the ripe stage, and analyzed. Each value was determined in three independent biological replicates and indicate a mean ± standard error (n = 3). Different lowercase letters indicate significant differences among fruit developmental stages in each tissue and different uppercase letters indicate significant differences among tissues at the ripe stage at P < 0.05 according to Tukey’s test.
Fig. 4. Effect of salt stress on soluble solids and serotonin content in fruit. Plants were supplied with 80 mM or 160 mM NaCl and fruit was harvested at the ripe stage. Pericarp tissues were used for the analysis and each value was determined in three independent biological replicates. Values indicate means ± standard error (n = 3). Values with the same letter were not significantly different at P < 0.05 according to Tukey’s test. The SSC data in control and 80 mM is the same as that in Ikeda et al. (2016).
Fig. 1. Serotonin content in fresh tomatoes. The nine cultivars or brands of tomatoes purchased and used for the analysis were cherry tomatoes (S1, S2, S3, S4), middle-sized tomatoes (M1, M2), and large-sized tomatoes (L1, L2 L3). SSC was measured for reference (A), as well as serotonin content (B). SSC for reference (C) and serotonin content (D) in fresh fruit were also measured for five and ten days at 4 ◦ C or 25 ◦ C, after the harvest at breaker stage. Pericarp tissues were used for the analysis and each value was determined in three independent biological replicates. Values indicate means ± standard error (n = 3). Values with the same letter were not significantly different at P < 0.05 according to Tukey’s test.
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the decarboxylation of tryptophan to form tryptamine, following which catalysis by tryptamine 5-hydroxylase (T5H) forms serotonin (Kang et al., 2009a; Park et al., 2009). In rice leaves, senescence increases TDC gene expression and serotonin content, and the overexpression of the TDC gene increases serotonin content and delays senescence (Kang et al., 2009a). Tryptamine synthesized by TDC is converted to serotonin by T5H, a cytochrome P450 enzyme (Fujiwara et al., 2010). Upon pathogen infection in pepper fruit, the expression of the TDC gene and serotonin content are increased to induce responses to infection (Park et al., 2009). Despite these previous findings, very few studies have focused on serotonin accumulation and the expression of serotonin synthesis-related genes during fruit development. In this study, to evaluate tomato fruit as a promising source of serotonin, we measured serotonin content in both fresh and processed tomatoes, and compared serotonin content in various fruit tissues. We also investigated serotonin synthesis-related genes to understand the determining factors of serotonin accumulation during fruit development.
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Fig. 2. Serotonin content in processed tomatoes. Tomato ketchup (K1, K2, K3), tomato juice (J1, J2), and canned whole tomatoes (W1, W2) were used for the analysis, and two or three items in each type of processed tomato were from different companies. Each value was determined in three independent extractions. Values indicate means ± standard error (n = 3). Values with the same letter were not significantly different at P < 0.05 according to Tukey’s test.
2. Materials and methods 2.1. Determination of serotonin content in fresh and processed tomatoes Fresh fruits of cherry tomatoes, middle-, and large-sized tomatoes were purchased from local markets, and their pericarp tissues
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Fig. 5. Alignment of amino acid sequences of SlTDC1, SlTDC2, and PepTDC1 (A) and the phylogenetic tree, based on the amino acid sequences of tryptophan decarboxylase (TDC) and tyrosine decarboxylase (TYDC) homologs (B). The alignment was constructed by Genetyx and ClustalW2 programs. Identical amino acids are denoted with black or gray. The conserved regions found in pyridoxal phosphate (PLP)-dependent decarboxylases are underlined. The arrow indicates a putative PLP-binding site (K). The tree was constructed by the neighbor-joining method, using the ClustalW2 program. Branch numbers refer to the percentage of replicates that support the branch, using the bootstrap method (1000 replicates). Species and accession numbers were as follows: OsTDC1 (Oryza sativa, AK069031), OsTDC3 (O. sativa, NM 001067504), SlTDC1 (S. lycopersicum, AK327027), SlTDC2 (S. lycopersicum, XM 004243286), PepTDC1
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Tomato ‘Ailsa Craig’ was grown in pots in a greenhouse according to the method of Ikeda et al. (2013), and exocarp, mesocarp, seed and jelly, placenta, and columella tissues were sampled at various stages of fruit development. For salt stress treatment, plants were supplied with 80 or 160 mM NaCl every week after flowering, as described previously (Yin et al., 2010). Pericarp tissues were sampled at the ripe stage. Soluble solids and serotonin content were measured as described in 2.1.
in large-sized tomatoes, although some of the differences were not statistically significant. In contrast, there was no significant difference in serotonin content between various sized tomatoes, except tomato M2 (Fig. 1B). Tomato M2 grown under abiotic stress conditions to increase SSC showed high serotonin content as well as high SSC. Once the tomatoes were harvested, the stability of serotonin was investigated for both 4◦ C and 25◦ C for a period of 10 days. As a result, serotonin content did not decrease during the period at either temperature, with SSC measured for reference (Fig. 1C, D). The stability of serotonin during storage for a similar period is supported by the previous study using banana (Adao and Gloria, 2005). Fruit ripening presumably progressed during the period because SSC increased. The following processed tomato products were used for evaluation of serotonin content (Fig. 2): 1) tomato ketchup (three brands); 2) tomato juice (two brands), and; 3) canned whole tomatoes (two brands). The serotonin content in juice was higher than that in ketchup and canned whole tomatoes; however, the average serotonin content in the processed tomato products was 0.06 mg g−1 FW, which was approximately a hundred times lower than that found in fresh tomatoes.
2.3. Sequence analysis of the TDC and T5H homologs in tomato
3.2. Serotonin accumulation during fruit development
Tomato sequences homologous to the pepper TDC gene (PepTDC1, Park et al., 2009) and the rice T5H gene (Fujiwara et al., 2010) were sought using the National Center for Biotechnology Information (NCBI) Basic Local Alignment Search Tool (BLAST) (http://www.ncbi.nlm.nih.gov/), and the three complementary DNA (cDNA) sequences found were named SlTDC1, SlTDC2, and SlT5H. Their sequences in the tomato genome were confirmed using the Sol Genomics Network (http://solgenomics.net/ ) database. Sequence alignment and phylogenetic trees were prepared using Genetyx software and Multiple Sequence Alignment by ClustalW2. A protein domain search was performed using InterProScan (http://www.ebi.ac.uk/interpro/).
An increase in serotonin content was observed during fruit development in all fruit tissues (Fig. 3). Serotonin content was lowest at 10 days after flowering (DAF) and was highest at the ripe stage. Serotonin content was very low in seed and jelly, placenta, and columella tissues at 10 and 20 DAF; yet, serotonin content increased remarkedly at 30 DAF. Serotonin content in mesocarp tissues was highest in various fruit tissues at the ripe stage, although differences in serotonin content were not large between tissues. Serotonin content in pericarp at the ripe stage increased under the stress condition of 160 mM NaCl when SSC was increased (Fig. 4).
were used for the analysis. Processed tomatoes, such as ketchup, juice, and canned whole tomatoes were also purchased from local markets. To evaluate changes in serotonin content during storage, ‘M82’ tomato plants were grown in a planting bed in a greenhouse according to the method of Ikeda et al. (2013), and breaker stage fruit were stored at 4◦ C and 25◦ C. Serotonin content was measured by the Serotonin EIA Kit (Beckman Coulter) according to the manufacturer’s instructions. Soluble solids content (SSC; Brix) in fresh tomatoes was measured for reference, using a Brix meter. 2.2. Determination of serotonin content during tomato fruit development
3.3. Sequence analysis of cDNA encoding SlTDC1, SlTDC2, and SlT5H
2.4. Expression analysis of SlTDC1, SlTDC2, and SlT5H Total RNA was extracted from fruit pericarp tissues at each stage, and removal of genomic DNA, reverse transcription, and real-time PCR were performed according to the methods of Mohammed et al. (2012). The following gene-specific primer sets were designed using the Primer 3 software: 5 -TAGTTCCGCAGCGTTCATTG-3 5 -CGAGTTTACGATCACGTGCAG-3 for SlTDC1; 5 and CGTTAAGTCCGATCCCATGT-3 and 5 -CTCAGTGAACGTCGCACCTA3 for SlTDC2; 5 -GTTGTCCAGGTTATGCTCTTGG-3 and 5 -CCTTCCCATGGCTACAATCAAC-3 for SlT5H; 5 and 5 -ACGTCCCTGACAATTTCACTGTATGCCAGTGGTCGT-3 CTCG-3 for SlActin (Accession No. BT012695). 3. Results 3.1. Serotonin contents of fresh and processed tomatoes Fresh tomatoes of various fruit sizes were used for the evaluation of serotonin content (Fig. 1). SSC, which is one of the most important qualities of tomato, was also determined for reference. SSC in cherry tomato S1 and S2 and middle-sized tomato M2 were significantly higher than that in large-sized tomatoes (Fig. 1A). SSCs in other cherry and middle-sized tomatoes were also higher than
Tomato sequences homologous to one of the pepper TDC genes (PepTDC1, Park et al., 2009) were sought using NCBI BLAST, resulting in two cDNA sequences being identified, which were named SlTDC1 and SlTDC2. It was also confirmed in the Sol Genomics Network database that two putative TDC genes exist in the tomato genome. The amino acid sequences of SlTDC1 and SlTDC2 were 85.7% and 83.3% identical to that of PepTDC1, respectively, whereas the SlTDC1 sequence was 89.3% identical to that of SlTDC2 (Fig. 5A). Using InterProScan, SlTDC1, and SlTDC2 were found to belong to aromatic amino acid decarboxylases, which are pyridoxal phosphate (PLP)-dependent decarboxylases. The conserved regions of PLP-dependent decarboxylases and PLP-binding sites were conserved in SlTDC1 and SlTDC2, as well as in PepTDC1 (Park et al., 2009). Tomato sequences homologous to another pepper TDC gene (PepTDC2) were also sought; however, no sequences showing good homology were found. A phylogenetic tree was constructed based on the amino acid sequences of TDC and tyrosine decarboxylase (TYDC), which belong to aromatic amino acid decarboxylases along with TDC (Fig. 5B). In the tree, SlTDC1, SlTDC2, and PepTDC1 fell into the TDC group, whereas PepTDC2 and OsTDC2 fell into the TYDC group. Tomato sequences homologous to the rice T5H gene (OsT5H, Fujiwara et al., 2010) were sought using NCBI BLAST, and a cDNA
(Capsicum annuum, FJ710789), PepTDC2 (C. annuum, FJ710788), CrTDC1 (Catharanthus roseus, M25151), OpTDC1 (Ophiorrhiza pumila, AB086168), CaTDC1 (Camptotheca acuminata, U73656), CaTDC2 (C. acuminata, U73657), AtTYDC (Arabidopsis thaliana, AY074539), OsTYDC (O. sativa, AK065830), OsTDC2 (O. sativa, AK103253), PsTYDC1 (Papaver somniferum, U08597), PsTYDC2 (P. somniferum, U08598), PcTYDC2 (Petroselinum crispum, M96070), and PcTYDC4 (P. crispum, M95685).
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Fig. 6. Alignment of amino acid sequences of SlT5H and OsT5H (A) and the phylogenetic tree, based on the amino acid sequences of SlT5H, OsT5H and tomato and rice proteins that belong to the cytochrome P450 groups close to the CYP71 group (B). The alignment was constructed by Genetyx and ClustalW2 programs. Identical amino acids are denoted with black. The K-helix motif and heme-binding motif are marked by dotted and thick underlines, respectively. The arrow indicates the glycine residue in the heme-binding motif, which is essential for T5H functioning (Fujiwara et al., 2010). The tree was constructed by the neighbor-joining method, using the ClustalW2 program. Branch numbers refer to the percentage of replicates that support the branch using the bootstrap method (1000 replicates). Species and accession numbers were as follows: OsT5H (O. sativa, AK071599), SlT5H (S. lycopersicum, NM 001247918). Other accession codes are from the cytochrome P450 homepage (http://drnelson.uthsc.edu/CytochromeP450.html).
sequence was identified, named SlT5H. It was confirmed in the Sol Genomics Network database that one putative T5H gene exists in the tomato genome. The amino acid sequence of SlT5H was 51.4% identical to that of OsT5H (Fig. 6A). According to Fujiwara et al. (2010) and the Sol Genomics Network database, OsT5H and SlT5H both belong to the CYP71 group of cytochrome P450. The K-helix motif and heme-binding motif were conserved in both SlT5H and OsT5H. The glycine residue in the heme-binding motif, which is essential for T5H function (Fujiwara et al., 2010), was also conserved in both SlT5H and OsT5H. A phylogenetic tree was constructed based on the amino acid sequences of SlT5H, OsT5H, and tomato and rice proteins belonging to the cytochrome P450 groups
close to the CYP71 group (Fig. 6B). Phylogenetically, SlT5H was most similar to OsT5H.
3.4. Comparison between changes in serotonin content and the SlTDC1, SlTDC2, and SlT5H mRNA levels during tomato fruit development The SlTDC1, SlTDC2, and SlT5H levels were measured in pericarp tissues during fruit development and compared with serotonin content (Fig. 7). Serotonin content increased until breaker stage and did not change thereafter. The SlTDC1 mRNA level increased until 35 DAF and then decreased. The SlTDC2 mRNA level was constantly
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Fig. 7. Changes in serotonin content (A) and the SlTDC1 (B), SlTDC2 (C), and SlT5H (D) mRNA levels during tomato fruit development. Serotonin content data (A) in 10, 20, 30 DAF, and Ripe is the same as that in Fig. 3 (mesocarp). Pericarp tissues were used for the analysis and each value was determined in three independent biological replicates. Data (B, C, D) shows the relative expression levels, normalized against SlActin. Values indicate means ± standard error (n = 3). Values with the same letter were not significantly different in each plot at P < 0.05 according to Tukey’s test.
very low, and was 4400 times lower than the SlTDC1 mRNA level at 35 DAF. The SlT5H mRNA remained at similar levels until the breaker stage, after which it decreased to the lowest level. 4. Discussion Serotonin contents were measured in fresh and processed tomatoes for comparison because knowledge of this comparison remains incomplete. Serotonin content was found to be very low in processed tomato products including juice, ketchup, and canned whole tomatoes. The results suggest that processing such as heat treatment might decrease serotonin content in tomatoes. Therefore, fresh tomatoes were a far better source of serotonin. The average serotonin content was 6.4 g g−1 FW in fresh tomatoes of various fruit sizes. This value of serotonin content was higher than that
of previous tomato recordings (3.2 g g−1 FW), and third-highest recorded in fresh fruits and vegetables (Feldman and Lee, 1985). It was also much higher than that recorded in other popular vegetables such as broccoli (0.2 g g−1 FW) and eggplant (0.2 g g−1 FW) (Feldman and Lee, 1985). Studies on serotonin accumulation in various fruit tissues throughout fruit development remain poorly represented in the literature. Serotonin content increased in all fruit tissues during fruit development and reached maximum levels at the ripe stage, when they are also usually the most palatable. Differences in serotonin content were relatively small among fruit tissues at the ripe stage, and serotonin content in mesocarp, which is the main fruit tissue, was highest among all fruit tissues. With regards to other functional food ingredients, differences among tomato fruit tissues in ascorbic acid content have been noted to be within a factor of two, whereas lycopene content has varied by a factor greater than ten (Moco et al., 2007). Our results and those of the previous study suggest that tissue localization of functional food ingredients varies. During storage, serotonin content was not reduced at either room temperature or the low temperature. Many beneficial components of tomato fruit have been shown to decrease during ripening and storage (Oms-Oliu et al., 2011), even though sugar content increases fractionally, as it also did in our study. These results suggest that fresh tomatoes are a promising source of serotonin. Generally, SSC corresponded to fruit size, except in one tomato type (M2) grown under abiotic stress to increase SSC. Serotonin content did not correspond to fruit size. Serotonin content in tomato fruit was also increased in our cultivation under salt stress conditions. Cultivation under abiotic stress conditions appears to be a promising method for producing high quality fruit, because in addition to serotonin production in the present study, the method may promote metabolism of sugar and other useful components such as amino acids (Ikeda et al., 2016). Tomato putative TDC genes, SlTDC1 and SlTDC2, were identified based on the amino acid sequence of PepTDC1 because PepTDC1 has been proven to be a TDC using the recombinant protein (Park et al., 2009). Sequence analysis confirmed that SlTDC1 and SlTDC2 belong to the TDC gene group. Two aromatic amino acid decarboxylase genes in pepper, PepTDC1 and PepTDC2 (Park et al., 2009), were classified into the TDC and TYDC gene groups, respectively, whereas both genes in tomato, SlTDC1 and SlTDC2, were classified into the TDC gene group. Tomato putative T5H gene, SlT5H, was also identified based on the amino acid sequence of OsT5H, because OsT5H has been proven to be a T5H, using the recombinant protein (Fujiwara et al., 2010). Sequence analysis confirmed that SlT5H belongs to the T5H gene group, i.e., the CYP71 group of cytochrome P450. Collectively, three genes that could be related to serotonin synthesis in tomato fruit were identified, and their expression was analyzed with serotonin accumulation. The SlTDC1 mRNA level increased with serotonin content during tomato fruit development, whereas the SlTDC2 mRNA level was constantly very low. SlTDC2 mRNA was also undetectable in the transcriptome analysis using custom DNA microarrays designed for the comprehensive array, consisting of probe sets based on the whole-genome sequence of tomato (Ikeda et al., 2016). The SlTDC2 mRNA was higher in leaves than in fruit (data not shown), suggesting its primary role in leaves rather than in fruit. A decrease in the SlTDC1 mRNA level after 35 DAF likely corresponds to the fact that serotonin content did not increase during the same period. Collectively, SlTDC1 might play a role in serotonin accumulation in tomato fruit. The SlT5H mRNA level was maintained from 15 DAF to breaker stage in tomato fruit, suggesting that SlT5H might play a role in serotonin synthesis from tryptamine, which SlTDC1 synthesized from tryptophan. TDC could be a key enzyme in serotonin synthesis
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in rice leaves, whereas the T5H gene is constitutively expressed (Kang et al., 2009a). Our results might support the previous study, although SlT5H could be the determinant of serotonin content in two tomato genotypes (Ikeda et al., 2016). Some studies on the physiological roles of serotonin in plants, such as in rice and pepper, found serotonin to be related to biotic and abiotic stress (Ishihara et al., 2008; Kang et al., 2009a; Park et al., 2009; Fujiwara et al., 2010). Serotonin increases from the normal level through stimulation by stress, and in these cases, to serve functions. Serotonin content in ripe tomato fruit in the present study was obviously higher than that recorded at the normal level in previous studies, and serotonin has high radical scavenging activity (Kang et al., 2009a). Therefore, the role of accumulated serotonin in tomato fruit is interestingly varied, and further studies will be necessary to clarify how its benefits can be optimized. In addition to serotonin, its metabolites such as melatonin and phenylpropanoid amides have also been reported to have physiological roles in biotic and abiotic stress responses in tomato (Kang et al., 2009b; Okazaki et al., 2009). Therefore, the accumulation of serotonin metabolites as well as serotonin should be investigated. The improvement of quality as well as the enhancement of stress tolerance have been proposed in pioneer work regarding metabolic engineering of tomato secondary metabolites (Davidovich-Rikanati et al., 2007), and the same type of study can be performed using SlTDC1. To the best of our knowledge, the present study is the first detailed study on serotonin accumulation during fruit development. The results revealed that fresh tomatoes are much more beneficial for serotonin intake than processed tomatoes, and serotonin accumulates during fruit development. We also suggest that SlTDC1, which is one of the tomato putative TDC family genes, as well as SlT5H, might play a role in the accumulation of serotonin. Further studies including transgenic experiments will be necessary to provide direct evidence for the functions of these enzymes because their functions were discussed using only sequence homology and expression data in this study. It is predicted that serotonin intake through daily consumption of vegetables and fruits can have anti-obesity effects, as it has been shown that daily consumption of banana, which similar to tomato and is rich in serotonin, increases serotonin in the bloodstream (Xiao et al., 1998). Our results could also be useful for the evaluation of tomato fruit quality in terms of serotonin content and the development of cultivation and breeding methods for serotonin-rich tomatoes. Acknowledgements The authors thank Hiroki Ikeda and Motoki Sato for their help and the National BioResource Project tomato (NBRP tomato) for information. This work was supported by the Research Project on Development of Agricultural Products and Foods with Health-promoting benefits (NARO) and Grants-in-Aid for Scientific Research [24248006]. References Adao, R.C., Gloria, M.B.A., 2005. Bioactive amines and carbohydrate changes during ripening of ‘Prata’ banana (Musa acuminata×M. balbisiana). Food Chem. 90, 705–711. Davidovich-Rikanati, R., Sitrit, Y., Tadmor, Y., Iijima, Y., Bilenko, N., Bar, E., Carmona, B., Fallik, E., Dudai, N., Simon, J.E., Pichersky, E., Lewinsohn, E.,2007.
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