Physiological roles of tryptophan decarboxylase revealed by overexpression of SlTDC1 in tomato

Abstract

SlTDC1, a candidate gene for tryptophan decarboxylase (TDC) in tomato, was the focus of this study because SlTDC1 may play a role in the biosynthesis of serotonin (Hano et al., 2017), which is a novel functional ingredient because of its anti-obesity effects; further, its developmental roles are largely unknown. Tomato was transformed with SlTDC1 (TDX lines) to demonstrate its enzymatic function, the developmental roles of SlTDC1 and serotonin, and the possibility of molecular breeding. Transformation increased serotonin concentration three times or more in fruit without growth inhibition; in contrast, the concentration of tryptophan, which is the substrate of sero-tonin biosynthesis, decreased. The results showed the roles of SlTDC1 and its usefulness in producing serotonin-rich fruits. In addition, our transgenic studies indicated that tryptamine 5-hydroxylase might be a key enzyme in serotonin biosynthesis. Curling leaf margins were observed in TDX lines, which may result from a decrease in tryptophan concentration. The number of days from flowering to the breaker ripening stage decreased in TDX fruit and wild-type fruit treated with serotonin, and the expression of ripening-related genes was promoted in TDX fruit in real-time PCR and RNA-sequencing analyses, indicating the role of serotonin in ripening. Collectively , our results revealed the horticultural importance of TDC in fruit with its biochemical and physiological roles.

Full text

Scientia Horticulturae 275 (2021) 109672
Available online 16 September 2020
0304-4238/© 2020 The Author(s). Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/4.0/).
Physiological roles of tryptophan decarboxylase revealed by overexpression
of SlTDC1 in tomato
Yui Tsunoda , Shohei Hano , Nozomi Imoto , Tomoki Shibuya
1
, Hiroki Ikeda
2
,
Kayoko Amagaya , Kazuhisa Kato, Hitoshi Shirakawa , Hisashi Aso , Yoshinori Kanayama *
Graduate School of Agricultural Science, Tohoku University, Aoba-ku, Sendai, 980-8572, Japan
ARTICLE INFO
Keywords:
Fruit
Ripening
Serotonin
Solanum lycopersicum
ABSTRACT
SlTDC1, a candidate gene for tryptophan decarboxylase (TDC) in tomato, was the focus of this study because
SlTDC1 may play a role in the biosynthesis of serotonin (Hano et al., 2017), which is a novel functional ingredient
because of its anti-obesity effects; further, its developmental roles are largely unknown. Tomato was transformed
with SlTDC1 (TDX lines) to demonstrate its enzymatic function, the developmental roles of SlTDC1 and serotonin,
and the possibility of molecular breeding. Transformation increased serotonin concentration three times or more
in fruit without growth inhibition; in contrast, the concentration of tryptophan, which is the substrate of sero-
tonin biosynthesis, decreased. The results showed the roles of SlTDC1 and its usefulness in producing serotonin-
rich fruits. In addition, our transgenic studies indicated that tryptamine 5-hydroxylase might be a key enzyme in
serotonin biosynthesis. Curling leaf margins were observed in TDX lines, which may result from a decrease in
tryptophan concentration. The number of days from owering to the breaker ripening stage decreased in TDX
fruit and wild-type fruit treated with serotonin, and the expression of ripening-related genes was promoted in
TDX fruit in real-time PCR and RNA-sequencing analyses, indicating the role of serotonin in ripening. Collec-
tively, our results revealed the horticultural importance of TDC in fruit with its biochemical and physiological
roles.
1. Introduction
Serotonin is an aromatic amine that is a well-known neurotrans-
mitter in the human central nervous system (Veenstra-VanderWeele
et al., 2000). However, approximately 98 % of serotonin is present in the
peripheral system, and this peripheral serotonin reportedly has an
anti-obesity effect (Watanabe et al., 2010). Since the concentration of
serotonin is high in tomato compared to other vegetables and fruits,
research on the novel function of serotonin in tomato has been initiated
(Hano et al., 2017). Tomato is a highly important solanaceous vegetable
crop. It is tasty and easily digestible and its bright color stimulates the
appetite. It is consumed as a salad with other leafy vegetables, in
sandwiches, and as a stewed, fried, and baked supplement separately, or
in combination with other vegetables (Sabijon and Sudaria, 2018; Khan
et al., 2019). Previously, serotonin concentrations have been shown to
be much higher in fresh fruit than processed tomato products and
increases during fruit development (Hano et al., 2017).
Two steps have been proposed as important in serotonin biosynthesis
in plants: the conversion of tryptophan to tryptamine by tryptophan
decarboxylase (TDC) and serotonin synthesis from tryptamine by
tryptamine 5-hydroxylase (T5H) (Kang et al., 2009; Kanjanaphachoat
et al., 2012; Hano et al., 2017). Furthermore, TDC has been suggested to
be a key enzyme in serotonin biosynthesis using transgenic rice plants.
Although expression analysis of candidate genes for TDC, SlTDC1, and
SlTDC2 and for T5H and SlT5H in tomato has been previously reported
(Hano et al., 2017), evidence of the function of these genes has not been
obtained using transgenic tomato plants.
In plants, serotonin may play a role in disease tolerance in rice,
bamboo, and pepper (Tanaka et al., 2003; Ishihara et al., 2008; Park
et al., 2009). In addition, abiotic stress, such as low temperature and
high salinity, increases serotonin (Gupta and De, 2017). Since serotonin
has antioxidant activity, reactive oxygen species likely are related to
Abbreviations: DEGs, differentially expressed genes; GO, gene ontology; TDC, tryptophan decarboxylase; T5H, tryptamine 5-hydroxylase.
* Corresponding author.
E-mail address: yoshinori.kanayama.a7@tohoku.ac.jp (Y. Kanayama).
1
Present address: Faculty of Life and Environmental Science, Shimane University, Matsue 690-8504, Japan.
2
Present address: School of Agriculture, Utsunomiya University, Moka 321-4415, Japan.
Contents lists available at ScienceDirect
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https://doi.org/10.1016/j.scienta.2020.109672
Received 17 January 2020; Received in revised form 1 June 2020; Accepted 19 August 2020

Scientia Horticulturae 275 (2021) 109672
2
these stress responses (Hayashi et al., 2016; Pelagio-Flores et al., 2016).
As described above, there have been several reports on the role of se-
rotonin in biotic and abiotic stress tolerance; however, little is known
about the involvement of serotonin in developmental physiology. In
Mimosa and Arabidopsis, serotonin may play a role in the regulation of
shoot organogenesis and root system architecture (Ramakrishna et al.,
2009; Pelagio-Flores et al., 2016). In contrast, there are conicting re-
ports on rice, i.e., inhibition of senescence (Kang et al., 2009) and pro-
motion of senescence with growth inhibition (Kanjanaphachoat et al.,
2012) in TDC-overexpressors.
In this study, tomato was transformed with SlTDC1, a candidate gene
for TDC that is mainly expressed in fruit (Hano et al., 2017; Pang et al.,
2018), to elucidate TDC function and to demonstrate that molecular
breeding of serotonin-rich fruits and vegetables is possible. Further, the
developmental roles of TDC and serotonin were revealed through
observation and analysis of overexpressors, in which early fruit ripening
was observed. On the other hand, since melatonin, which is associated
with serotonin metabolism, has been previously reported to promote
fruit ripening (Sun et al., 2015), the concentration of melatonin was
determined in addition to serotonin in this study.
2. Materials and methods
2.1. Plant materials
Full-length cDNA of SlTDC1 (LEFL2019I23) was obtained from the
National BioResource Project-Tomato (http://tomato.nbrp.jp) and used
in PCR with high delity Taq enzyme. The following gene specic
primers, containing the attB sites, for PCR were designed: attB1 forward
(5′-GGGGACAAGTTTGTACAAAAAAGCAGGCTTCGAAGGAGATA-
GAACCATGGGAAGCCTTGATTCCAATAAC-3′) and attB2 reverse (5′-
GGGGACCACTTTGTACAAGAAAGCTGGGTCTTAAAACA-
CACTTTTTCTCAGCAAA-3′). Gateway technology (Thermo Fisher Sci-
entic) was used in cloning the amplied SlTDC1 gene into a pDONR
expression vector. The gene in the entry clone vector was then cloned
into a destination vector, pB-OX-GW, containing the CaMV 35S pro-
moter (Inplanta Innovations Inc.). Using the resulting plasmid, trans-
formation of tomato cultivar Micro-Tom was performed by Inplanta
Innovations Inc. (Sun et al., 2006).
Transgenic tomato overexpressing SlTDC1 (TDX line) at the T
0
gen-
eration was transplanted into pots lled with Sumi-Soil (Sumika Agro-
tech Co., Ltd.) and grown at 25 ◦C under an articial light 16-h
photoperiod (with PPDF of 100
μ
mol m
−2
s
−1
). The plants were sup-
plemented with nutrient solution (Hyponex Japan) every week. The
introduction of the transgene and normal ploidy of transformants
(Cytotechs) were assessed, and expression analysis was performed to
select transgenic plants with high expression of SlTDC1. Homozygotes of
three selected TDX lines, TDX3, TDX5, and TDX6, at the T
2
generation
were grown as described for the T
0
generation and used for observation
and analysis. Azygotes were used as a control. Pericarp at the breaker
stage and mature leaves at 120 days after gemination were harvested
and stored at −80 ◦C for gene expression and metabolite analyses.
Arabidopsis thaliana was also transformed to express the SlTDC1
(ATDX line) and was analyzed as part of supplementary data. For
A. thaliana (Col-0) transformation, a tomato transformation vector was
used by Inplanta Innovation Inc. The plants were grown as described
above in a vermiculite–pearlite (1:1) mixture. The transformed seeds
(T
1
) were selected using a medium containing kanamycin. For further
selection, a transgene check and an expression analysis were performed
using PCR. The homozygotes of three ATDX lines selected in the T
3
generation were analyzed.
2.2. Analysis of gene expression and metabolites
RNA was extracted using the RNeasy Plant Mini Kit (Qiagen). cDNA
was prepared and real-time PCR was performed following the methods
of Ikeda et al. (2016). Primer sequences for SlTCD1 and SlT5H were from
Hano et al. (2017) and those for SlACO1, SlASC2, SlASC4, and SlCNR
were from Sun et al. (2015) and Manning et al. (2006). Primer sequences
for SlActin as a control were from Mohammed et al. (2012).
Fluorescence-detection HPLC analysis of serotonin, tryptophan, and
tryptamine was performed following the procedures of Islam et al.
(2016). Melatonin concentration was determined using the Melatonin
ELISA Kit (Enzo Life Sciences, Inc.).
2.3. Tryptophan and serotonin treatment
Wild-type cultivar Micro-Tom was grown as described above and
used for serotonin treatment. Fruits were dipped in 1 mM, 10 mM, and
50 mM serotonin solution containing 0.01 % (v/v) Tween 20 for 5 s 3
times per week after the rst owers opened. TDX3 and TDX5 lines were
grown as described above and used for tryptophan treatment. After the
rst owers opened, 1 mM, 10 mM, and 50 mM tryptophan solution
containing 0.01 % (v/v) Tween 20 were sprayed on plants 3 times per
week.
Fig. 1. SlTDC1 mRNA levels in the fruit (a) and leaves (b) of TDX lines and
SlT5H mRNA levels in the fruit of TDX lines (c). Each value was determined in
three independent biological replicates. Data shows the relative expression
levels, normalized against SlActin. Values indicate means ±standard error
(n =3). Values with ** were signicantly different from control at P <0.01
according to Dunnett’s lest.
Y. Tsunoda et al.

Scientia Horticulturae 275 (2021) 109672
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2.4. RNA-sequencing (RNA-seq) and data analysis
Thirty days after owering, fruits from the TDX3 and control lines
were used for RNA-seq analysis. Three biological replicates were used in
the analysis. A sequencing library was generated using the TruSeq
Stranded mRNA LT Sample Prep Kit, and sequencing was performed
using the NovaSeq 6000 System with the NovaSeq 6000 S4 Reagent Kit
(Illumina). Artifacts, such as low quality reads, adaptor sequences, DNA
contamination, and PCR duplicates, were removed, and trimmed reads
were mapped to a reference genome with HISAT2 (https://ccb.jhu.edu/
software/hisat2/index.shtml) and assembled with StringTie (https://cc
b.jhu.edu/software/stringtie/). Expression proles were represented as
read counts and normalization values, which are based on transcript
lengths and depth of coverage. The Fragments Per Kilobase of transcript
per Million Mapped reads (FPKM) value was used as a normalization
value. Differentially expressed transcripts were ltered out through
statistical hypothesis testing (|fold change| ≥2, P <0.05 with t-tests),
and functional annotation and gene-set enrichment analysis of differ-
entially expressed genes (DEGs) were performed using the DAVID tool
(http://david.abcc.ncifcrf.gov/) based on the Gene Ontology (GO; http:
//geneontology.org/) and KEGG (http://www.genome.jp/kegg/)
databases.
Fig. 2. Serotonin (a), tryptamine (b), tryptophan (c), and melatonin (d) con-
centrations in the fruit of TDX lines. Each value was determined in three in-
dependent biological replicates. Values indicate means ±standard error (n =3).
Values with ** are signicantly different from the control at P <0.01 according
to Dunnett’s lest.
Fig. 3. Serotonin (a), tryptamine (b), and tryptophan (c) concentrations in the
leaves of TDX lines. Each value was determined in three independent biological
replicates. Values indicate means ±standard error (n =3). Values with ** are
signicantly different from the control at P <0.01 according to Dunnett’s lest.
Y. Tsunoda et al.

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3. Results
3.1. SlTDC1 mRNA levels and the concentrations of serotonin,
tryptamine, and tryptophan in TDX and ATDX lines
The levels of SlTDC1 mRNA in fruit and leaves were higher in TDX
lines than in the control (Fig. 1a, b). In fruit, serotonin and tryptamine
concentrations were higher in TDX lines than in the control; in contrast,
the concentration of tryptophan, which is a substrate for serotonin
biosynthesis, was lower in TDX lines than in the control (Fig. 2a–c). The
melatonin concentration in fruit was similar between TDX and control
lines, although it was only slightly higher in TDX6 than in the control
(Fig. 2d). In leaves, serotonin and tryptamine concentrations were
higher in TDX lines than in the control; in contrast, tryptophan con-
centration was lower in TDX lines than in the control (Fig. 3a–c). To
conrm the role of SlTDC1 in serotonin synthesis, SlTDC1 was expressed
in A. thaliana (ATDX lines). The expression of SlTDC1 in ATDX lines was
conrmed, and the serotonin and tryptamine concentrations were
higher in ATDX lines than in the control (Fig. S1a–c). There was no
signicant difference between the tryptophan concentrations of ATDX
lines and the wild-type A. thaliana plants (Fig. S1d). The serotonin
concentration in the wild-type plants and ATDX lines was considerably
lower than that of tomato.
3.2. Growth of TDX lines
The number of nodes to rst owers and the rate of fruit set did not
differ signicantly between TDX lines and the control (Fig. 4a, b). The
length of main stems and the number of owers did not differ signi-
cantly between TDX lines and control with the exception of one TDX line
(Fig. 4c, d). In contrast, the number of days from owering to breaker
stage was less in TDX3 and TDX5, which had high serotonin concen-
tration in fruit, than in the control, and the ratio of leaves with curled
leaf margins to total leaves was higher in TDX lines than in the control
(Fig. 4e, f).
3.3. Tryptophan and serotonin treatment
Since early ripening and leaf curling were observed in TDX lines,
tryptophan and serotonin treatment was performed. The number of days
from owering to breaker stage was less in 10 mM and 50 mM treat-
ments than in the control for serotonin treatment of wild-type fruit
(Fig. 5a). Serotonin concentrations in the fruit treated with serotonin
increased with serotonin treatment concentrations (Fig. 5b). Leaf curl-
ing was not observed in wild-type plants treated with serotonin, and
there was reduced leaf curling in TDX lines treated with 10 mM and
50 mM tryptophan (Fig. S2).
Fig. 4. Growth of TDX lines. The number of
nodes (a) and fruit set (b) indicate the number
of nodes to rst inorescences and fruit set
ratio, respectively. Main stem length (c) and the
number of owers (d) indicate the length of
main stems to rst inorescences and the
number of owers per plant, respectively. Days
to breaker (e) indicates the number of days from
owering to breaker stage in each fruit. Leaf
margin curling (f) indicate the percentage of the
number of curled leaves to total leaves. Values
indicate means ±standard error (n =9 to 29 in
a, b, c, d, and f; n =43 to 106 in e). Values with
** and * are signicantly different from the
control at P <0.01 and P <0.05, respectively,
according to Dunnett’s lest.
Y. Tsunoda et al.

Scientia Horticulturae 275 (2021) 109672
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3.4. Expression analysis of ripening-related genes
Since early ripening was observed in TDX fruit and serotonin-treated
fruit, expression analysis of ripening-related genes was performed in
TDX lines. In fruit 30 days after owering, the mRNA levels of SlACO1,
SlACS2, and SlACS4 were higher in TDX lines than in the control, and
mRNA levels of SlCNR were higher in TDX3 and TDX5 than in the
control (Fig. 6a–d).
3.5. RNA-seq analysis in TDX and control fruits
To further evaluate the early ripening phenotype of TDX fruit, RNA-
seq analysis was performed. cDNA libraries were generated from TDX3
and control fruits. From each cDNA library, approximately 50 million
sequence reads were obtained, resulting in approximately 5 Gb of
sequence data (Table S1). Among the total genes detected in TDX3 and
control lines using FPKM >1, 1,267 genes (P <0.05) were found to be
DEGs between TDX and control fruits, with 663 up-regulated and 604
down-regulated genes (Fig. S3). Hierarchical clustering showed clear
differences in expression proles between TDX3 and control lines
(Fig. S4). The potential function of DEGs was examined using GO
enrichment analysis. Enrichment data based on biological processes are
shown in Table 1, which clearly indicates the ripening-related functions
of the DEGs. The DEGs were primarily enriched in ethylene biosynthesis
and fruit ripening; the expression of genes, which play roles in ethylene
biosynthesis and cell wall metabolism, associated with the GO terms
were up-regulated as shown in Table 2. Up-regulation of ripening-
related genes and down-regulation of photosynthesis-related genes
were observed for other GO terms in Table 1. The DEGs enriched in cutin
biosynthesis were down-regulated, supporting the observation that the
cuticle formed in the tomato fruit epidermis before the ripe stage
(Giménez et al., 2015).
4. Discussion
Serotonin concentrations were approximately three times higher or
more in the fruit and leaves of TDX lines, which were overexpressors of
SlTDC1, compared to the control. Concentrations of tryptophan, which
is the substrate in serotonin biosynthesis, were lower in the fruit and
leaves of TDX lines compared to the control. The results indicate that
SlTDC1 functions as TDC and plays a key role in serotonin biosynthesis.
Indeed, SlTDC1 mRNA level increases with serotonin accumulation
during fruit development in wild-type tomato (Hano et al., 2017). Se-
rotonin concentration also increased with the overexpression of the TDC
Fig. 5. Days to breaker (a) and serotonin concentration (b) in wild-type fruit
treated with serotonin. Days to breaker indicates the number of days from
owering to breaker stage in each fruit. Values indicate means ±standard error
(n =11 to 21 in a; n =3 in b). Values with ** and * are signicantly different
from the control at P <0.01 and P <0.05, respectively, according to Dun-
nett’s lest.
Fig. 6. The mRNA levels of ripening-related genes in the fruit of TDX lines. The
expression of ethylene-related genes, SlACO1 (a), SlACS2 (b), and SlACS4 (c),
and their upstream gene SlCNR (d) was analyzed in fruit 30 days after ow-
ering. Each value was determined in three independent biological replicates.
Data show the relative expression levels, normalized against SlActin. Values
indicate means ±standard error (n =3). Values with ** are signicantly
different from the control at P <0.01 according to Dunnett’s lest.
Y. Tsunoda et al.

Scientia Horticulturae 275 (2021) 109672
6
gene in rice (Kang et al., 2009). Moreover, growth was not inhibited in
TDX lines. Therefore, the TDC gene may represent a potential target for
molecular breeding to increase the concentration of serotonin as a novel
functional ingredient with anti-obesity effects. To our knowledge, this is
the rst report of a signicant increase in serotonin concentration in
fruit.
The levels of SlTDC1 mRNA in fruit and leaves were the highest in
TDX3, followed by TDX6. Serotonin concentration in leaves corre-
sponded to the levels of SlTDC1 mRNA; in contrast, serotonin concen-
tration in fruit was highest in TDX3, followed by TDX5 and did not
correspond to SlTDC1 mRNA levels. Further, concentration of trypt-
amine, which is the direct product of TDC, in fruit was highest in TDX3,
followed by TDX6 and corresponded to SlTDC1 mRNA levels. Moreover,
expression analysis of SlT5H, which converts tryptamine to serotonin,
revealed that the level of SlT5H mRNA was lower in TDX6 than in TDX3
and TDX5, according to Tukey’s test (Fig. 1c). The results suggest that
the lowest concentration of serotonin in TDX6 fruit is related to the T5H
step of biosynthesis. TDC has been proposed as a key enzyme in sero-
tonin biosynthesis in rice (Kang et al., 2009), and our results also showed
its importance in tomato; however, T5H could be a key rate-limiting
factor in serotonin biosynthesis in tryptamine-rich tissues.
To conrm this, Arabidopsis thaliana, which has a low serotonin
concentration, was transformed with SlTDC1 (Fig. S1). As a result,
tryptamine accumulated in the transgenic Arabidopsis at 10 to 25
μ
g g
−1
FW, which was the same order of magnitude as tryptamine concentra-
tion in tomato; however, the increase in serotonin concentration was
minimal, i.e., two orders of magnitude lower than in tomato. The small
increase in the serotonin concentration was probably due to its origi-
nally low concentration in A. thaliana although the increase rates were
similar between serotonin and tryptamine. These ndings could be
important in considering molecular breeding of serotonin and the
physiological role of this metabolic pathway. However, for the fruit of
wild-type tomato, TDC is likely the determinant of serotonin concen-
tration, based on the correlation between serotonin concentration and
gene expression (Table S2; Catal´
a et al., 2017).
Since curling in leaf margins was observed in TDX lines, wild-type
plants were treated with serotonin to investigate its cause; however,
plants treated with serotonin did not have curling in the leaf margins.
Next, TDX plants were treated with tryptophan because the concentra-
tion of tryptophan decreased with an increase in serotonin concentration
in TDX leaves compared to the control. As a result, leaf margin curling
was recovered by the addition of tryptophan. The results suggest that the
leaf curling is owing to a decrease in tryptophan concentration rather
than an increase in serotonin concentration. Since auxin and serotonin
biosynthesis have the common precursor tryptophan, stress-induced
inhibition of root growth could be owing to partial impairment of
auxin functions from increased serotonin biosynthesis (Mukherjee et al.,
2014). Further, similar leaf deformation has been observed in tomato
plants in which auxin transport has been impaired by RNA interference
(Pattison and Catal´
a, 2012). Therefore, TDC seems to be an important
branch point in tryptophan metabolism.
In rice, opposite ndings have been reported for the role of serotonin,
i.e., inhibition (Kang et al., 2009) and promotion (Kanjanaphachoat
et al., 2012) of senescence. In this study, vegetative growth was not
inuenced, and yellowing owing to senescence was not observed in TDX
lines. In contrast, while tomato plants treated with 1 mM and 10 mM
serotonin grew normally, browning and death were observed in plants
treated with 50 mM serotonin. This nding is consistent with results for
Arabidopsis treated with serotonin (Kanjanaphachoat et al., 2012). In
addition, this previous work reported that tryptamine did not have the
same effect as serotonin. Although serotonin may play a role in stress
tolerance as stated in the introduction section (Hayashi et al., 2016;
Pelagio-Flores et al., 2016), serotonin at concentrations above a certain
threshold seems to promote senescence.
The number of days from owering to breaker stage was less in TDX
fruit and serotonin-treated fruit than in the control, suggesting that se-
rotonin promotes fruit ripening. Since the concentration of melatonin,
which can be synthesized from serotonin (Okazaki et al., 2009), some-
what increases during fruit development (Okazaki and Ezura, 2009) and
melatonin treatment promotes fruit ripening (Sun et al., 2015) in to-
mato, melatonin concentration was determined in TDX fruit. Melatonin
did not increase in TDX lines in which fruit ripening was enhanced,
indicating that the promotion of ripening in TDX fruit is not related to
melatonin. Early ripening in TDX fruit was conrmed using real-time
PCR by the enhanced expression of SlACO1, SlACS2, and SlACS4,
which play roles in ethylene production during tomato fruit ripening
(Klee and Giovannoni, 2011). RNA-seq analysis was also performed to
evaluate the early ripening phenotype in TDX fruit. More than a thou-
sand genes were found to be DEGs between TDX and control fruits in the
RNA-seq analysis, and the enrichment analysis revealed the early
ripening of TDX fruit. From real-time PCR (Fig. 6), the expression of
SlCNR, which functions upstream of ethylene-related genes (Liu et al.,
2015), was shown to increase in TDX fruit. The expression pattern of
SlCNR, with mRNA level increasing during fruit development prior to
ripening, is similar to the accumulation pattern of serotonin (Liu et al.,
2015; Hano et al., 2017), suggesting that serotonin may play a role in
increasing the expression of SlCNR and its downstream genes. This hy-
pothesis should be examined in subsequent research.
5. Concluding remarks
Tomato was transformed with SlTDC1 to demonstrate the enzymatic
function of SlTDC1, developmental roles of SlTDC1 and serotonin, and
explore the possibility of molecular breeding of serotonin-rich fruits.
The transformation increased the serotonin concentration in the fruits
without growth inhibition. These results demonstrate the roles of
SlTDC1 and its application in producing serotonin-rich fruits. The
Table 1
Enrichment analysis of DEGs based on Gene Ontology (Biological process).
Enrichment analysis was performed for 1,267 DEGs (Fig. S3) and the top 10
terms based on the P value are shown.
Accession Description Fold
enrichment
P value
GO:0009693 ethylene biosynthetic process 9.85 0.00018
GO:0009835 fruit ripening 9.03 0.00029
GO:0018298 protein-chromophore linkage 4.28 0.00092
GO:0009768 photosynthesis, light harvesting in
photosystem I
4.86 0.00246
GO:0009416 response to light stimulus 3.28 0.00290
GO:0009407 toxin catabolic process 4.01 0.00309
GO:0010143 cutin biosynthetic process 7.52 0.00319
GO:0009992 cellular water homeostasis 3.95 0.00730
GO:0034220 ion transmembrane transport 3.95 0.00730
GO:0009813 avonoid biosynthetic process 2.20 0.00772
Table 2
Fold changes in expression levels in genes involved in the "ethylene biosynthetic
process" and "fruit ripening" in Table 1.
Entrez gene
ID
Product Fold change (TDX/
Control)
100125909 1-aminocyclopropane-1-carboxylate
oxidase
2.21
101268031 1-aminocyclopropane-1-carboxylate
oxidase homolog
4.10
544052 1-aminocyclopropane-1-carboxylate
oxidase 1
2.85
544285 ethylene-forming enzyme 3.40
606304 1-aminocyclopropane-1-carboxylate
synthase 2
5.86
778356 1-aminocyclopropane-1-carboxylate
synthase
2.32
544051 polygalacturonase-2a 5.69
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Scientia Horticulturae 275 (2021) 109672
7
curling leaf margins observed in the TDX lines may result from a
decrease in the tryptophan concentration. The number of days from
owering to breaker ripening stage decreased in the TDX and wild-type
fruits treated with serotonin, indicating the role of serotonin in ripening.
Taken collectively, our results reveal the horticultural importance of
TDC and its biochemical and physiological roles.
CRediT authorship contribution statement
Yui Tsunoda: Investigation, Writing - original draft. Shohei Hano:
Investigation. Nozomi Imoto: Investigation, Writing - original draft.
Tomoki Shibuya: Methodology, Formal analysis, Data curation. Hiroki
Ikeda: Methodology, Formal analysis, Resources. Kayoko Amagaya:
Investigation. Kazuhisa Kato: Resources, Writing - review & editing,
Project administration. Hitoshi Shirakawa: Methodology, Resources,
Supervision. Hisashi Aso: Conceptualization, Funding acquisition.
Yoshinori Kanayama: Conceptualization, Writing - review & editing,
Supervision, Project administration, Funding acquisition.
Declaration of Competing Interest
The authors report no declarations of interest.
Acknowledgements
The authors thank the National BioResource Project tomato (NBRP
tomato) for bioresource and information. This research was supported
by the Research Project on Development of Agricultural Products and
Foods with Health-promoting benets (NARO) and Grants-in-Aid for
Scientic Research [24248006].
Appendix A. Supplementary data
Supplementary material related to this article can be found, in the
online version, at doi:https://doi.org/10.1016/j.scienta.2020.109672.
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