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Citation: Lee, K.; Back, K. Functional
Characterization of the Ciliate
Stylonychia lemnae Serotonin
N-Acetyltransferase, a Pivotal
Enzyme in Melatonin Biosynthesis
and Its Overexpression Leads to
Peroxidizing Herbicide Tolerance in
Rice. Antioxidants 2024,13, 1177.
https://doi.org/10.3390/
antiox13101177
Academic Editors: Marina
Garcia-Macia and Adrián
Santos-Ledo
Received: 4 September 2024
Revised: 25 September 2024
Accepted: 25 September 2024
Published: 27 September 2024
Copyright: © 2024 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
antioxidants
Article
Functional Characterization of the Ciliate Stylonychia lemnae
Serotonin N-Acetyltransferase, a Pivotal Enzyme in Melatonin
Biosynthesis and Its Overexpression Leads to Peroxidizing
Herbicide Tolerance in Rice
Kyungjin Lee and Kyoungwhan Back *
Department of Molecular Biotechnology, College of Agriculture and Life Sciences, Chonnam National University,
Gwangju 61186, Republic of Korea; nicekj7@hanmail.net
*Correspondence: kback@chonnam.ac.kr; Tel.:+82-62-530-2165
Abstract: Serotonin N-acetyltransferase (SNAT) is a pivotal enzyme for melatonin biosynthesis
in all living organisms. It catalyzes the conversion of serotonin to N-acetylserotonin (NAS) or 5-
methoxytrypytamine (5-MT) to melatonin. In contrast to animal- and plant-specific SNAT genes,
a novel clade of archaeal SNAT genes has recently been reported. In this study, we identified
homologues of archaeal SNAT genes in ciliates and dinoflagellates, but no animal- or plant-specific
SNAT homologues. Archaeal SNAT homologue from the ciliate Stylonychia lemnae was annotated as a
putative N-acetyltransferase. To determine whether the putative S. lemnae SNAT (SlSNAT) exhibits
SNAT enzyme activity, we chemically synthesized and expressed the full-length SlSNAT coding
sequence (CDS) in Escherichia coli, from which the recombinant SlSNAT protein was purified by
Ni
2+
affinity column chromatography. The recombinant SlSNAT exhibited SNAT enzyme activity
toward serotonin (K
m
= 776
µ
M) and 5-MT (K
m
= 246
µ
M) as substrates. Furthermore, SlSNAT-
overexpressing (SlSNAT-OE) transgenic rice plants showed higher levels of melatonin synthesis
than wild-type controls. The SlSNAT-OE rice plants exhibited delayed leaf senescence and tolerance
against treatment with the reactive oxygen species (ROS)-inducing herbicide butafenacil by decreasing
hydrogen peroxide (H
2
O
2
) and malondialdehyde (MDA) levels, suggesting that melatonin alleviates
ROS production in vivo.
Keywords: archaea; ciliophoran; melatonin; Osl20; serotonin N-acetyltransferase; Stylonichia lemnae;
transgenic rice
1. Introduction
Organisms from all kingdoms of life synthesize melatonin [
1
]. First discovered in 1958,
melatonin was identified as a pineal factor that lightened melanocytes [
2
,
3
]. Although its
skin-lightening effects were restricted to amphibians, many other biological activities have
been documented, among which the best-described roles are in the regulation of the sleep–
wake cycle and cellular redox homeostasis in animals [
4
,
5
]. Additionally, melatonin plays
key roles in survival by orchestrating protein quality control, such as the chaperone network,
autophagy, and the ubiquitin–proteasome system, in both animals and
plants [6–8]
; it also
acts as a potent antioxidant by scavenging a range of harmful oxidants [9].
Melatonin is biosynthesized through a four-step sequential enzymatic reaction with
tryptophan as the first substrate in all organisms [
10
,
11
]. The common last intermediate
is serotonin, produced by two enzymes—tryptophan hydroxylase (TPH) and aromatic
amino acid decarboxylase (AADC) in animals, and tryptophan decarboxylase (TDC) and
tryptamine 5-hydroxylase (T5H) in plants. As for the last two enzymatic reactions, arylalky-
lamine N-acetyltransferase (AANAT) (also designated as SNAT) and N-acetylserotonin O-
methyltransferase (ASMT) are involved in melatonin biosynthesis. These enzymes catalyze
Antioxidants 2024,13, 1177. https://doi.org/10.3390/antiox13101177 https://www.mdpi.com/journal/antioxidants
Antioxidants 2024,13, 1177 2 of 13
the conversion of serotonin to either N-acetylserotonin (NAS) or 5-methoxytryptamine
(5-MT) in both animals and plants. NAS is synthesized by SNAT, whereas 5-MT is syn-
thesized by ASMT, followed by melatonin synthesis through the action of SNAT. NAS
leads to melatonin synthesis by ASMT. Interestingly, NAS is converted back to serotonin by
the action of NAS deacetylase in plants, resulting in the accumulation of serotonin (rather
than melatonin) [
12
]. Therefore, the pathway from serotonin
→
5-MT
→
melatonin can
overcome the reverse reaction of classical melatonin biosynthesis (serotonin
→
NAS
→
melatonin), which leads to enhanced melatonin production, as observed in plants exposed
to various stressors [13].
Among the four enzymes, SNAT is thought to play pivotal roles because it is closely
associated with both rhythmic melatonin synthesis in animals [
14
] and the physiological
functions of melatonin in plants [
15
]. Correspondingly, a range of SNAT genes have been
cloned from numerous animal and plant species, although there is no apparent amino acid
sequence identity between animal and plant SNAT genes [
11
]. SNAT exists as a single
copy in animals, whereas plants harbor at least two copies [
16
]. Surprisingly, a substantial
portion of NAS synthesis occurs in an AANAT-independent manner in hamsters and rats,
suggesting the existence of an alternative SNAT enzyme, such as protein N-acetyltransferase
(NAT) [
17
]. Consistent with the predictions of Slominski [
17
], it was recently reported
that human Naa50, a NAT family protein, exhibited SNAT enzyme activity [
18
]. Human
Naa50 can catalyze the conversion of both serotonin and 5-MT into NAS and melatonin,
similar to animal and plant SNAT proteins [
16
,
19
]. The successful cloning of an alternative
SNAT from humans, Naa50, was attributed to the cloning of archaeon SNAT because they
are functional orthologues [
18
,
20
]. Unlike the animal-specific AANAT and plant-specific
SNAT genes, human Naa50 or archaeal SNAT orthologues are distributed in all kingdoms
of life, including ciliates and dinoflagellates, because melatonin is ubiquitously present
throughout nature. For example, the ciliate Tetrahymena pyriformis and dinoflagellate
Gonyaulax polyedra are representative species that synthesize melatonin, but neither AANAT
nor SNAT orthologues have been identified in their genomes [1].
Here, we found many human Naa50 orthologues in ciliates and dinoflagellates; the
ciliate Stylonychia lemnae Naa50 orthologue was annotated as a putative NAT (GenBank
accession number CDW73552). To determine whether the protein product of the Naa50
orthologue from S. lemnae exhibits SNAT enzyme activity, we expressed the putative S.
lemnae SNAT (SlSNAT) in Escherichia coli, from which the recombinant SlSNAT protein
was purified and subjected to analysis of SNAT enzyme kinetics
in vitro
. Furthermore,
we performed
in vivo
functional analysis by transforming the SlSNAT gene into rice to
determine whether its ectopic overexpression was coupled with melatonin biosynthesis in
transgenic plants along with enhanced tolerance to oxidative stress.
2. Materials and Methods
2.1. Codon-Optimized Chemical Synthesis of S. lemnae SNAT Gene
Based on the amino acid sequence information of S. lemnae SNAT (GenBank accession
number CDW73552), the full-length nucleotides of S. lemnae SNAT with the length of 546 bp
were codon optimized by reference of rice SNAT2 codon and custom synthesized (Bioneer,
Daejeon, Republic of Korea).
2.2. Escherichia coli Expression, Production, and Recombinant S. lemnae SNAT
Protein Purification
Expression, production, and recombinant protein purification of SlSNAT have been
described previously elsewhere [
18
,
21
]. In brief, the full-length synthetic SlSNAT gene
was amplified by PCR by using a primer set (SlSNAT forward primer, 5
′
-ACC ATG GCC
ATG CCG GCG CCC GAG GCG-3
′
;SlSNAT reverse primer, 5
′
-CTC GAG CTG CGA CGT
GGT CGA CTG-3
′
) with a template plasmid containing the synthetic SlSNAT DNA such as
pBHA-SlSNAT which was synthesized by Bioneer. The PCR product was ligated into the
TA vector (RBC Bioscience, New Taipei City, Taiwan) followed by plasmid purification of
Antioxidants 2024,13, 1177 3 of 13
TA-SlSNAT. The TA-SlSNAT plasmid was digested with NcoI and XhoI restriction enzymes.
Then, the NcoI and XhoI insert of SlSNAT DNA was ligated into the same restriction
sites of the E. coli expression vector pET32b (Novagen, Madison, WI, USA) to construct
the pET32b-SlSNAT vector construct. As for pET300-SlSNAT vector construction, the
full-length SlSNAT DNA of 546 nucleotides in length was amplified by PCR using a
primer set (forward primer 5
′
-AAA AAG CAG GCT CCA TGC CGG CGC CCG AGG-
3
′
; reverse primer 5
′
-AGA AAG CTG GGT CTA CTG CGA CGT GGT CGA-3
′
) using
the synthetic SlSNAT DNA as the template. The resulting PCR product was further
amplified by PCR using an attB primer set [
18
]. The full-length SlSNAT PCR product was
cloned into the pDONR221 gateway vector (Invitrogen, Carlsbad, CA, USA) via the BP
recombination reaction. The pDONR221-SlSNAT gene entry vector was then recombined
with the pET300 destination vector (Invitrogen) via LR recombination to yield pET300-
SlSNAT. Both the pET32b-SlSNAT and pET300-SlSNAT plasmids were transformed into E.
coli strain BL21(DE3) (Invitrogen, Carlsbad, CA, USA). Further E. coli culture, isopropyl-
β
-D-thiogalactopyranoside (IPTG; Sigma, St. Louis, MO, USA) treatment, and affinity
(Ni
2+
) purification were described in detail previously [
21
]. The purified thioredoxin (Trx)-
tagged SlSNAT fusion protein was mixed with the equal volume of glycerol and stored at
−
80
◦
C until use. Protein concentrations were determined using the Bradford method and
a protein assay dye (Bio-Rad, Hercules, CA, USA).
2.3. Homology and Phylogenetic Analysis
The analysis of amino acid sequence homology search using human Naa50 as a query
was carried out with the BLASTp tool in the non-redundant protein sequences databases
at the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/,
accessed on 26 August 2019). Phylogenetic tree analysis was achieved by using the BLAST-
Explorer program (version 2, Information Genomique & Structurale, Marseille, France) [
22
].
2.4. Enzymatic Assays for SNAT
The enzymatic assay for SNAT was performed in a 100
µ
L final volume containing
0.8
µ
g of the purified recombinant Trx-SlSNAT protein, 0.5 mM serotonin (or other sub-
strates), and 0.5 mM acetyl-CoA in 100 mM potassium phosphate (pH 8.8 or varying pH)
at 55 ◦C (or other temperatures) for 30 min. Twenty microliters of enzymatic reaction was
analyzed by reverse phase high-performance liquid chromatography (HPLC) as described
previously [23]. The Lineweaver–Burk plots were employed to calculate substrate affinity
(K
m
) and the maximum reaction rate (V
max
) using two substrates such as serotonin and
5-methoxytryptamine. The analysis was performed in triplicate.
2.5. Generation of Transgenic Rice Plants Overexpressing the Synthetic SlSNAT Gene
In order to deliver the synthetic SlSNAT DNA into the rice genome, we utilized a
pIPKb002 gateway binary vector. The pIPKb002 binary vector was kindly provided by
Dr. J. Kumlehn (Leibniz Institute of Plant Genetics and Crop Plant Search, Gatersleben,
Germany) [
24
]. In brief, the pDONR221-SlSNAT gene entry vector was then recombined
with the pIPKb002 destination vector via LR recombination to yield pIPKb002-SlSNAT,
which was then transformed into Agrobacterium tumefaciens strain LBA4404. Agrobacterium-
mediated rice transformation using the calli generated from a japonica rice cultivar called
Dongjin was employed to generate transgenic rice plants as described previously [25].
2.6. Melatonin Measurement from the SlSNAT Overexpression (SlSNAT-OE) Transgenic
Rice Plants
Melatonin levels were measured in frozen rice leaf samples (0.1 g) using the TissueL-
yser II (Qiagen, Tokyo, Japan). Melatonin was quantified by high-performance liquid
chromatography (HPLC) with a fluorescence detector system (Waters, Milford, MA, USA)
as described previously [18].
Antioxidants 2024,13, 1177 4 of 13
2.7. Senescence Treatment in the SlSNAT-OE Transgenic Rice Plants
Rice leaves from rice grown in soil for 5 weeks in a glass house at 28
◦
C under a
12 h light/12 h dark cycle at a photosynthetic photon flux density of 150
µ
mol m
−2
s
−1
were detached for an
in vitro
senescence experiment. Groups of 10 segments (detached
fourth and fifth leaves) were transferred to 50 mL polypropylene conical tubes containing
25 mL of water. The samples were incubated for 12 d under the same growth conditions as
described above. The entire rice leaves were frozen in liquid nitrogen and pulverized to a
powder using a TissueLyser II instrument (Qiagen). As for the measurement of chlorophyll
contents, the powder (100 mg) was extracted with 1 mL of 0.1 M NH
4
OH (containing
80% acetone). Chlorophyll concentrations were determined at wavelengths of 647, 644,
and 750 nm using a spectrophotometer (Optizen POP-Bio; Mecasys, Daejeon, Republic of
Korea) according to Porra et al. [
26
]. The levels of malondialdehyde (MDA) were measured
at wavelengths of 440, 532, and 600 nm using a spectrophotometer (Optizen POP-Bio) as
described previously [27].
2.8. Total RNA Isolation and Reverse Transcription–Polymerase Chain Reaction (RT-PCR)
Total RNA was isolated using a Ribospin Plant Kit (GeneAll Biotechnology Co., Seoul,
Republic of Korea). RT-PCR was conducted using a rice ubiquitin-5 gene (UBQ5) as the
loading control. The sequences of primers were listed previously [
27
]. As for the real-time
PCR analysis, a Mic qPCR Cycler system (Bio Molecular Systems, Coomera, Queensland,
VIC, Australia) with the Luna Universal qPCR Master Mix (New England Biolabs, Ipswich,
MA, USA) was utilized. The expression of genes was analyzed using Mic’s RQ software
v2.2 (Bio Molecular Systems) and normalized to UBQ5 as described previously [28].
2.9. Tolerance against Peroxidizing Herbicide Butafenacil
Surface-sterilized dehusked rice seeds were sown on half-strength Murashige and
Skoog (MS) medium [
27
]. The 7-day-old seedlings collected from MS medium were
incubated in 50 mL polypropylene conical tubes containing 0.1
µ
M butafenacil (a kind gift
from Dr. Guh (Chonnam National University, Gwangju, Republic of Korea)) for 12 h in
the dark followed by a 12 h light/12 h dark cycle for 48 h. Cellular leakage in medium
was determined using a conductivity meter (Cole-Parmer Instrument LLC, IL, USA) as
described previously [
29
]. Hydrogen peroxide contents were quantified by an OxiTec™
Hydrogen Peroxide/Peroxidase (H2O2) Assay Kit (Biomax, Guri-si, Republic of Korea).
2.10. Statistical Analysis
The data were analyzed by analysis of variance using IBM SPSS Statistics 23 software
(IBM Corp. Armonk, NY, USA) as described previously [27].
3. Results
3.1. Selection and Chemical Synthesis of Stylonychia lemnae SNAT Gene
Analysis using BLASTp (http://www.ncbi.nlm.nih.gov/, accessed on 26 August 2019)
indicated that human Naa50 harboring SNAT enzyme activity [
18
] exhibited ~38% identity
to a putative SNAT protein of S. lemnae consisting of 181 amino acids (aa) with 83% query
cover value. Phylogenetic analysis indicated that the putative S. lemnae SNAT (SlSNAT)
protein was the closest orthologue of human Naa50 belonging to the archaeal SNAT clade
(Figure 1A). The two protein sequences were aligned using BLASTp, revealing that SlSNAT
had 38% aa identity to human Naa50 (Figure 1B). The putative SlSNAT was annotated
as a member of the protein NAT family carrying a region with identity to E. coli RimI,
ranging in size from 40 to 176 aa. It was recently reported that RimI, an N-terminal
protein acetyltransferase, also exhibited SNAT enzyme activity [
21
]. Taken together, the
results of these in silico analyses suggested that the putative SlSNAT may exhibit SNAT
enzyme activity.
Antioxidants 2024,13, 1177 5 of 13
Antioxidants 2024, 13, x FOR PEER REVIEW 5 of 14
(SlSNAT) protein was the closest orthologue of human Naa50 belonging to the archaeal
SNAT clade (Figure 1A). The two protein sequences were aligned using BLASTp, reveal-
ing that SlSNAT had 38% aa identity to human Naa50 (Figure 1B). The putative SlSNAT
was annotated as a member of the protein NAT family carrying a region with identity to
E. coli RimI, ranging in size from 40 to 176 aa. It was recently reported that RimI, an N-
terminal protein acetyltransferase, also exhibited SNAT enzyme activity [21]. Taken to-
gether, the results of these in silico analyses suggested that the putative SlSNAT may ex-
hibit SNAT enzyme activity.
Figure 1. (A) Phylogenetic tree of Stylonichia lemnae SNAT and archaeal ortholog genes. The scale
bar represents 0.4 substitutions per site. S. lemnae SNAT is wrien in bold for emphasis. (B) Amino
acid sequence identity and similarity between S. lemnae SNAT and human Naa50 (SNAT). The con-
served acetyl-coenzyme-A-binding sites are underlined. Dashes denote gaps. GenBank accession
numbers are archaea SNAT (NC_002689), E. coli RimI (WP_137442509), human Naa50 (BAB14397),
rice SNAT3 (AK241100), and S. lemnae SNAT (CDW73552).
To verify its function, we first synthesized the full-length coding sequence (CDS) of
the SlSNAT gene in accordance with rice SNAT2 codon usage (GenBank accession number
AK068156) for efficient SlSNAT gene expression in rice plants. The complete 546 nucleo-
tides of the synthetic SlSNAT CDS are shown in Figure 2A. Changing the third codon
position from A or T to G or C increased the G+C content of synthetic SlSNAT. Therefore,
a total of 133 of 182 codons were modified. As expected, the G+C content of synthetic SlS-
NAT increased to 59% (Figure 2B), in contrast to 38% for native SlSNAT, and was therefore
much closer to that of rice SNAT2 (70%).
Figure 1. (A) Phylogenetic tree of Stylonichia lemnae SNAT and archaeal ortholog genes. The scale bar
represents 0.4 substitutions per site. S. lemnae SNAT is written in bold for emphasis. (B) Amino acid
sequence identity and similarity between S. lemnae SNAT and human Naa50 (SNAT). The conserved
acetyl-coenzyme-A-binding sites are underlined. Dashes denote gaps. GenBank accession numbers
are archaea SNAT (NC_002689), E. coli RimI (WP_137442509), human Naa50 (BAB14397), rice SNAT3
(AK241100), and S. lemnae SNAT (CDW73552).
To verify its function, we first synthesized the full-length coding sequence (CDS) of
the SlSNAT gene in accordance with rice SNAT2 codon usage (GenBank accession number
AK068156) for efficient SlSNAT gene expression in rice plants. The complete 546 nucleotides
of the synthetic SlSNAT CDS are shown in Figure 2A. Changing the third codon position
from A or T to G or C increased the G+C content of synthetic SlSNAT. Therefore, a total
of 133 of 182 codons were modified. As expected, the G+C content of synthetic SlSNAT
increased to 59% (Figure 2B), in contrast to 38% for native SlSNAT, and was therefore much
closer to that of rice SNAT2 (70%).
Antioxidants 2024, 13, x FOR PEER REVIEW 6 of 14
Figure 2. (A) Nucleotide alignment between native (red; CDW73552) and synthetic (blue) S. lemnae
SNAT. Identity is denoted by stars. Black leers, amino acids. (B) Modification of S. lemnae SNAT
codons. The nucleotide sequence of synthetic S. lemnae SNAT was manually codon optimized with
reference to the rice SNAT2 codon.
3.2. Purification of Recombinant SlSNAT and Enzyme Kinetic Analysis
The synthetic full-length SlSNAT CDS was first cloned into pET300 for expression
with an N-terminal hexa-histidine tag and purified by Ni
2+
affinity column chromatog-
raphy. However, this recombinant SlSNAT protein was insoluble and could not be puri-
fied (Figure 3A). To enhance solubility, we used a thioredoxin (Trx)-tagged SlSNAT ex-
pression system employing the pET32b vector. The soluble Trx-SlSNAT recombinant pro-
tein was purified by Ni
2+
affinity column chromatography, although the majority of the
expressed protein remained insoluble (Figure 3A). The purified recombinant Trx-SlSNAT
protein was first examined for SNAT enzyme activity in catalyzing the conversion of ser-
otonin to NAS. As shown in Figure 3B, the recombinant SlSNAT exhibited SNAT-specific
enzyme activity of 7.1 pkat/mg protein, which was similar to the activity reported previ-
ously for an archaeon SNAT (6.7 pkat/mg protein) [20]. The SNAT enzyme activity of SlS-
NAT was 1.8-fold higher than that of E. coli RimI [21] but 4.7-fold lower than that of rice
SNAT3 [28]. All SNAT enzymes from animals or plants can accept many other amines as
substrates [16,30]. The highest SNAT enzyme activity was observed with the tyramine
substrate, followed in order by serotonin, 5-MT, and tryptamine (Figure 3B). Both ar-
chaeon SNAT and rice SNAT3 showed the highest SNAT enzyme activity toward tyramine
as a substrate, whereas E. coli RimI showed preference for 5-MT over other amines. The
biological significance of substrate preference among SNAT enzymes has not been eluci-
dated. Based on these observations, SlSNAT was confirmed to exhibit SNAT enzyme ac-
tivity. These results suggested that S. lemnae can directly synthesize melatonin in the pres-
ence of 5-MT and indirectly synthesize melatonin via NAS. Consistent with mechanisms
observed in other organisms, S. lemnae can synthesize melatonin by two pathways: from
serotonin to NAS and melatonin, and from serotonin to 5-MT and melatonin.
Figure 2. (A) Nucleotide alignment between native (red; CDW73552) and synthetic (blue) S. lemnae
SNAT. Identity is denoted by stars. Black letters, amino acids. (B) Modification of S. lemnae SNAT
codons. The nucleotide sequence of synthetic S. lemnae SNAT was manually codon optimized with
reference to the rice SNAT2 codon.
Antioxidants 2024,13, 1177 6 of 13
3.2. Purification of Recombinant SlSNAT and Enzyme Kinetic Analysis
The synthetic full-length SlSNAT CDS was first cloned into pET300 for expression
with an N-terminal hexa-histidine tag and purified by Ni
2+
affinity column chromatogra-
phy. However, this recombinant SlSNAT protein was insoluble and could not be purified
(Figure 3A). To enhance solubility, we used a thioredoxin (Trx)-tagged SlSNAT expression
system employing the pET32b vector. The soluble Trx-SlSNAT recombinant protein was
purified by Ni
2+
affinity column chromatography, although the majority of the expressed
protein remained insoluble (Figure 3A). The purified recombinant Trx-SlSNAT protein was
first examined for SNAT enzyme activity in catalyzing the conversion of serotonin to NAS.
As shown in Figure 3B, the recombinant SlSNAT exhibited SNAT-specific enzyme activity of
7.1 pkat/mg protein, which was similar to the activity reported previously for an archaeon
SNAT (6.7 pkat/mg protein) [
20
]. The SNAT enzyme activity of SlSNAT was 1.8-fold higher
than that of E. coli RimI [
21
] but 4.7-fold lower than that of rice SNAT3 [
28
]. All SNAT
enzymes from animals or plants can accept many other amines as substrates [
16
,
30
]. The
highest SNAT enzyme activity was observed with the tyramine substrate, followed in order
by serotonin, 5-MT, and tryptamine (Figure 3B). Both archaeon SNAT and rice SNAT3
showed the highest SNAT enzyme activity toward tyramine as a substrate, whereas E.
coli RimI showed preference for 5-MT over other amines. The biological significance of
substrate preference among SNAT enzymes has not been elucidated. Based on these obser-
vations, SlSNAT was confirmed to exhibit SNAT enzyme activity. These results suggested
that S. lemnae can directly synthesize melatonin in the presence of 5-MT and indirectly
synthesize melatonin via NAS. Consistent with mechanisms observed in other organisms,
S. lemnae can synthesize melatonin by two pathways: from serotonin to NAS and melatonin,
and from serotonin to 5-MT and melatonin.
Antioxidants 2024, 13, x FOR PEER REVIEW 7 of 14
Figure 3. Escherichia coli expression, affinity purification of SlSNAT recombinant protein, and its en-
zymatic characteristics. (A) Expression of SlSNAT as a thioredoxin (Trx) fusion protein using a
pET32b vector and expression of SlSNAT as an N-terminal His × 6-tagged SlSNAT protein using a
pET300 vector. (B) Serotonin N-acetyltransferase enzyme activity (SNAT) as a function of various
substrates. The expression of recombinant SlSNAT protein is marked by arrows.
Similar to other SNAT enzymes, such as plant SNAT, human Naa50, and E. coli RimI,
the optimal temperature and pH of SlSNAT toward serotonin as a substrate were 55 °C
and pH 7.8, respectively (Figure 4A,B) [16,18,21]. The Km and Vmax values of SlSNAT to-
ward serotonin as a substrate were 776 µM and 1.47 nmol/min/mg protein, respectively
(Figure 4C). For 5-MT as a substrate, SlSNAT exhibited Km and Vmax values of 246 µM and
0.362 nmol/min/mg protein, respectively (Figure 4D). The catalytic efficiency (Vmax/Km)
was slightly higher toward serotonin than toward 5-MT, suggesting that SlSNAT shows
substrate preference for serotonin over 5-MT during melatonin biosynthesis. However, in
the presence of lower substrate concentrations, SlSNAT preferentially utilized 5-MT dur-
ing melatonin synthesis because it showed higher substrate affinity for 5-MT than for ser-
otonin. Further in-depth studies are required to determine whether SlSNAT exhibits pro-
tein NAT activity similar to that of human Naa50 [18].
Figure 4. SNAT enzyme kinetic analysis. Serotonin N-acetyltransferase enzyme activity as a function
of (A) various temperature, (B) various pH, (C) Km and Vmax values for serotonin substrate, (D) Km
and Vmax values for 5-methoxytryptamine (5-MT) substrate. Values are means ± SD (n = 3). nd, not
detectable.
Figure 3. Escherichia coli expression, affinity purification of SlSNAT recombinant protein, and its
enzymatic characteristics. (A) Expression of SlSNAT as a thioredoxin (Trx) fusion protein using a
pET32b vector and expression of SlSNAT as an N-terminal His
×
6-tagged SlSNAT protein using a
pET300 vector. (B) Serotonin N-acetyltransferase enzyme activity (SNAT) as a function of various
substrates. The expression of recombinant SlSNAT protein is marked by arrows.
Similar to other SNAT enzymes, such as plant SNAT, human Naa50, and E. coli RimI,
the optimal temperature and pH of SlSNAT toward serotonin as a substrate were 55
◦
C
and pH 7.8, respectively (Figure 4A,B) [
16
,
18
,
21
]. The K
m
and V
max
values of SlSNAT
toward serotonin as a substrate were 776
µ
M and 1.47 nmol/min/mg protein, respectively
(Figure 4C). For 5-MT as a substrate, SlSNAT exhibited K
m
and V
max
values of 246
µ
M and
0.362 nmol/min/mg protein, respectively (Figure 4D). The catalytic efficiency (V
max
/K
m
)
was slightly higher toward serotonin than toward 5-MT, suggesting that SlSNAT shows
substrate preference for serotonin over 5-MT during melatonin biosynthesis. However, in
the presence of lower substrate concentrations, SlSNAT preferentially utilized 5-MT during
melatonin synthesis because it showed higher substrate affinity for 5-MT than for serotonin.
Further in-depth studies are required to determine whether SlSNAT exhibits protein NAT
activity similar to that of human Naa50 [18].
Antioxidants 2024,13, 1177 7 of 13
Antioxidants 2024, 13, x FOR PEER REVIEW 7 of 14
Figure 3. Escherichia coli expression, affinity purification of SlSNAT recombinant protein, and its en-
zymatic characteristics. (A) Expression of SlSNAT as a thioredoxin (Trx) fusion protein using a
pET32b vector and expression of SlSNAT as an N-terminal His × 6-tagged SlSNAT protein using a
pET300 vector. (B) Serotonin N-acetyltransferase enzyme activity (SNAT) as a function of various
substrates. The expression of recombinant SlSNAT protein is marked by arrows.
Similar to other SNAT enzymes, such as plant SNAT, human Naa50, and E. coli RimI,
the optimal temperature and pH of SlSNAT toward serotonin as a substrate were 55 °C
and pH 7.8, respectively (Figure 4A,B) [16,18,21]. The Km and Vmax values of SlSNAT to-
ward serotonin as a substrate were 776 µM and 1.47 nmol/min/mg protein, respectively
(Figure 4C). For 5-MT as a substrate, SlSNAT exhibited Km and Vmax values of 246 µM and
0.362 nmol/min/mg protein, respectively (Figure 4D). The catalytic efficiency (Vmax/Km)
was slightly higher toward serotonin than toward 5-MT, suggesting that SlSNAT shows
substrate preference for serotonin over 5-MT during melatonin biosynthesis. However, in
the presence of lower substrate concentrations, SlSNAT preferentially utilized 5-MT dur-
ing melatonin synthesis because it showed higher substrate affinity for 5-MT than for ser-
otonin. Further in-depth studies are required to determine whether SlSNAT exhibits pro-
tein NAT activity similar to that of human Naa50 [18].
Figure 4. SNAT enzyme kinetic analysis. Serotonin N-acetyltransferase enzyme activity as a function
of (A) various temperature, (B) various pH, (C) Km and Vmax values for serotonin substrate, (D) Km
and Vmax values for 5-methoxytryptamine (5-MT) substrate. Values are means ± SD (n = 3). nd, not
detectable.
Figure 4. SNAT enzyme kinetic analysis. Serotonin N-acetyltransferase enzyme activity as a function
of (A) various temperature, (B) various pH, (C)K
m
and V
max
values for serotonin substrate, (D)K
m
and V
max
values for 5-methoxytryptamine (5-MT) substrate. Values are means
±
SD (n = 3). nd,
not detectable.
3.3. Transgenic Rice Plants Overexpressing SlSNAT
To investigate the biological function of SlSNAT, ectopic overexpression of SlSNAT
in rice was performed under the control of the constitutive maize ubiquitin promoter. A
total of 19 independent transgenic lines were generated through Agrobacterium-mediated
transformation. Six homozygous lines of T
2
seeds were further selected to examine the
gain-of-function effects of SlSNAT expression on melatonin synthesis in rice. To confirm the
ectopic overexpression of the SlSNAT transgene mRNA, reverse transcription polymerase
chain reaction (RT-PCR) analysis was performed in the SlSNAT-overexpressing (SlSNAT-
OE) transgenic rice plants. SlSNAT mRNA was detected in total RNA isolated from
the leaves of 7-day-old transgenic rice seedlings. All except one transgenic line (line 15)
exhibited a high level of transgene expression, whereas no detectable SlSNAT mRNA was
observed in wild-type controls (Figure 5A).
To determine whether SlSNAT overexpression was functionally associated with mela-
tonin synthesis, the levels of melatonin were measured in 7-day-old seedlings of SlSNAT-OE
lines. As shown in Figure 5B, all transgenic lines (with the exception of line 15) produced
higher levels of melatonin than wild-type control, indicating a positive correlation between
SlSNAT mRNA expression and melatonin level. It was previously reported that rice SNAT2
downregulation resulted in short grains, in conjunction with a decrease in brassinosteroid
(BR) level [
31
]. As a simple phenotypic test for increased BR level, grain length was first
monitored in the SlSNAT-OE lines. As shown in Figure 5C, some transgenic lines (e.g., lines
7, 18, and 19) exhibited a slight increase in grain length, whereas other lines (e.g., lines 5, 10,
and 15) showed similar grain length to the wild-type control. These results indicated that
BR levels were not significantly increased in the SlSNAT-OE lines relative to the wild type.
Lamina angle (the angle between the second leaf blade and vertical culm) was monitored
as another BR indicator because BR plays a key role in lamina angle increase. As shown
in Figure 5D,E, the lamina angles of the SlSNAT-OE lines were comparable to those of
wild-type controls, indicating that BR levels were not elevated in the SlSNAT-OE lines;
Antioxidants 2024,13, 1177 8 of 13
this finding was similar to previous results from transgenic rice plants overexpressing rice
SNAT2 [
31
]. Taken together, these observations indicated that an endogenous melatonin
increase is not necessarily coupled to an increase in BR.
Antioxidants 2024, 13, x FOR PEER REVIEW 8 of 14
3.3. Transgenic Rice Plants Overexpressing SlSNAT
To investigate the biological function of SlSNAT, ectopic overexpression of SlSNAT
in rice was performed under the control of the constitutive maize ubiquitin promoter. A
total of 19 independent transgenic lines were generated through Agrobacterium-mediated
transformation. Six homozygous lines of T
2
seeds were further selected to examine the
gain-of-function effects of SlSNAT expression on melatonin synthesis in rice. To confirm
the ectopic overexpression of the SlSNAT transgene mRNA, reverse transcription poly-
merase chain reaction (RT-PCR) analysis was performed in the SlSNAT-overexpressing
(SlSNAT-OE) transgenic rice plants. SlSNAT mRNA was detected in total RNA isolated
from the leaves of 7-day-old transgenic rice seedlings. All except one transgenic line (line
15) exhibited a high level of transgene expression, whereas no detectable SlSNAT mRNA
was observed in wild-type controls (Figure 5A).
Figure 5. Generation of SlSNAT overexpression transgenic rice and the melatonin content of rice
seedlings. (A) RT-PCR analyses of transgenic and wild-type 7-day-old rice seedlings. (B) Melatonin
contents of 7-day-old rice seedlings. (C) Photograph of seed length. (D) Photograph of lamina angle
in 3-week-old rice seedling. (E) Measurement of lamina angle. WT, wild type; UBQ5, rice ubiquitin
5 gene. GenBank accession number of UBQ5 is AK061988. Different leers indicate significant dif-
ferences among lines (Tukey’s HSD; p < 0.05).
To determine whether SlSNAT overexpression was functionally associated with mel-
atonin synthesis, the levels of melatonin were measured in 7-day-old seedlings of SlSNAT-
OE lines. As shown in Figure 5B, all transgenic lines (with the exception of line 15) pro-
duced higher levels of melatonin than wild-type control, indicating a positive correlation
between SlSNAT mRNA expression and melatonin level. It was previously reported that
rice SNAT2 downregulation resulted in short grains, in conjunction with a decrease in
brassinosteroid (BR) level [31]. As a simple phenotypic test for increased BR level, grain
length was first monitored in the SlSNAT-OE lines. As shown in Figure 5C, some trans-
genic lines (e.g., lines 7, 18, and 19) exhibited a slight increase in grain length, whereas
other lines (e.g., lines 5, 10, and 15) showed similar grain length to the wild-type control.
These results indicated that BR levels were not significantly increased in the SlSNAT-OE
lines relative to the wild type. Lamina angle (the angle between the second leaf blade and
vertical culm) was monitored as another BR indicator because BR plays a key role in lam-
ina angle increase. As shown in Figure 5D,E, the lamina angles of the SlSNAT-OE lines
were comparable to those of wild-type controls, indicating that BR levels were not ele-
vated in the SlSNAT-OE lines; this finding was similar to previous results from transgenic
rice plants overexpressing rice SNAT2 [31]. Taken together, these observations indicated
that an endogenous melatonin increase is not necessarily coupled to an increase in BR.
Figure 5. Generation of SlSNAT overexpression transgenic rice and the melatonin content of rice
seedlings. (A) RT-PCR analyses of transgenic and wild-type 7-day-old rice seedlings. (B) Melatonin
contents of 7-day-old rice seedlings. (C) Photograph of seed length. (D) Photograph of lamina angle
in 3-week-old rice seedling. (E) Measurement of lamina angle. WT, wild type; UBQ5, rice ubiquitin
5 gene. GenBank accession number of UBQ5 is AK061988. Different letters indicate significant
differences among lines (Tukey’s HSD; p< 0.05).
3.4. Elevated Melatonin Levels Confer Senescence Tolerance
Melatonin acts as a potent antioxidant that scavenges many oxidants, including reac-
tive oxygen species (ROS) and reactive nitrogen species (RNS) [
9
]. Aging and senescence
are major physiological consequences of a lack of antioxidant activity in both animals
and plants [
32
,
33
]. To determine whether SlSNAT-OE transgenic rice plants exhibited
senescence tolerance mediated by increased melatonin levels, detached rice leaves were
subjected to senescence treatment.
As shown in Figure 6A, the SlSNAT-OE lines exhibited delayed leaf senescence,
indicated by increased chlorophyll levels compared with wild-type control (Figure 6B). In
parallel with the increased chlorophyll levels, the contents of malondialdehyde (MDA), one
of the end products of lipid peroxidation, were decreased in the SlSNAT-OE lines relative
to wild type, indicating that the SlSNAT-OE lines displayed oxidative stress tolerance
(Figure 6C). Consistent with the biochemical data highlighting senescence tolerance, several
senescence marker genes (e.g., Osl2,Osl20, and Osl185 [
34
]) were expressed at lower
levels in the SlSNAT-OE lines than in wild-type controls, as determined by RT-PCR and
quantitative RT-PCR (qRT-PCR) (Figure 6D,E). Taken together, these data showed that
the elevated endogenous melatonin levels mediated by SlSNAT overexpression in the
transgenic rice plants conferred tolerance against senescence. This was mainly attributed to
the enhanced synthesis of melatonin, a potent antioxidant that efficiently scavenges ROS.
Antioxidants 2024,13, 1177 9 of 13
Antioxidants 2024, 13, x FOR PEER REVIEW 9 of 14
3.4. Elevated Melatonin Levels Confer Senescence Tolerance
Melatonin acts as a potent antioxidant that scavenges many oxidants, including re-
active oxygen species (ROS) and reactive nitrogen species (RNS) [9]. Aging and senescence
are major physiological consequences of a lack of antioxidant activity in both animals and
plants [32,33]. To determine whether SlSNAT-OE transgenic rice plants exhibited senes-
cence tolerance mediated by increased melatonin levels, detached rice leaves were sub-
jected to senescence treatment.
As shown in Figure 6A, the SlSNAT-OE lines exhibited delayed leaf senescence, in-
dicated by increased chlorophyll levels compared with wild-type control (Figure 6B). In
parallel with the increased chlorophyll levels, the contents of malondialdehyde (MDA),
one of the end products of lipid peroxidation, were decreased in the SlSNAT-OE lines
relative to wild type, indicating that the SlSNAT-OE lines displayed oxidative stress tol-
erance (Figure 6C). Consistent with the biochemical data highlighting senescence toler-
ance, several senescence marker genes (e.g., Osl2, Osl20, and Osl185 [34]) were expressed
at lower levels in the SlSNAT-OE lines than in wild-type controls, as determined by RT-
PCR and quantitative RT-PCR (qRT-PCR) (Figure 6D,E). Taken together, these data
showed that the elevated endogenous melatonin levels mediated by SlSNAT overexpres-
sion in the transgenic rice plants conferred tolerance against senescence. This was mainly
aributed to the enhanced synthesis of melatonin, a potent antioxidant that efficiently
scavenges ROS.
Figure 6. Enhanced senescence tolerance in SlSNAT-overexpressing transgenic rice plants. (A) Pho-
tograph of senescence-treated 5-week-old rice leaves. (B) Chlorophyll contents in senescence-treated
rice leaves. (C) Malondialdehyde (MDA) contents. (D) Gene expression profiles of senescence
marker genes by RT-PCR. (E) Gene expression profiles of senescence marker genes by quantitative
RT-PCR. Fourth and fifth leaves from 5-week-old rice plants grown in soil pots were detached and
this was followed by senescence treatment for 12 days. WT, wild type; UBQ5, rice ubiquitin 5 gene.
GenBank accession numbers are Osl2 (AF251073), Osl20 (AF251067), Osl185 (AF251075), and UBQ5
(AK061988). Different leers indicate significant differences among the lines (Tukey’s HSD; p < 0.05).
3.5. Melatonin Confers Tolerance against the Peroxidizing Herbicide Butafenacil
Butafenacil is a peroxidizing herbicide that targets protoporphyrinogen oxidase in-
volved in chlorophyll biosynthesis, resulting in massive production of ROS followed by
cell death [35]. Due to the potent antioxidant activity of melatonin, it was expected that
SlSNAT-OE lines would exhibit tolerance against butafenacil. As shown in Figure 7, the
Figure 6. Enhanced senescence tolerance in SlSNAT-overexpressing transgenic rice plants. (A) Photo-
graph of senescence-treated 5-week-old rice leaves. (B) Chlorophyll contents in senescence-treated
rice leaves. (C) Malondialdehyde (MDA) contents. (D) Gene expression profiles of senescence marker
genes by RT-PCR. (E) Gene expression profiles of senescence marker genes by quantitative RT-PCR.
Fourth and fifth leaves from 5-week-old rice plants grown in soil pots were detached and this was
followed by senescence treatment for 12 days. WT, wild type; UBQ5, rice ubiquitin 5 gene. GenBank
accession numbers are Osl2 (AF251073), Osl20 (AF251067), Osl185 (AF251075), and UBQ5 (AK061988).
Different letters indicate significant differences among the lines (Tukey’s HSD; p< 0.05).
3.5. Melatonin Confers Tolerance against the Peroxidizing Herbicide Butafenacil
Butafenacil is a peroxidizing herbicide that targets protoporphyrinogen oxidase in-
volved in chlorophyll biosynthesis, resulting in massive production of ROS followed by
cell death [
35
]. Due to the potent antioxidant activity of melatonin, it was expected that
SlSNAT-OE lines would exhibit tolerance against butafenacil. As shown in Figure 7, the
SlSNAT-OE lines showed herbicide tolerance as indicated by reduced levels of cellular
leakage, MDA, and H
2
O
2
production compared with the wild-type controls. These observa-
tions suggested that enhanced endogenous melatonin production is closely associated with
oxidative stress tolerance upon exposure to the peroxidizing herbicide butafenacil. Similar
results were also observed in transgenic rice seedlings overexpressing sheep SNAT [36].
Antioxidants 2024, 13, x FOR PEER REVIEW 10 of 14
SlSNAT-OE lines showed herbicide tolerance as indicated by reduced levels of cellular
leakage, MDA, and H
2
O
2
production compared with the wild-type controls. These obser-
vations suggested that enhanced endogenous melatonin production is closely associated
with oxidative stress tolerance upon exposure to the peroxidizing herbicide butafenacil.
Similar results were also observed in transgenic rice seedlings overexpressing sheep SNAT
[36].
Figure 7. Enhanced tolerance of SlSNAT-overexpressing transgenic rice plants against peroxidizing
herbicide butafenacil. (A) Photograph of rice seedlings after butafenacil treatment. (B) Effect of bu-
tafenacil treatment on cellular leakage. (C) MDA production from butafenacil-treated rice seedlings.
(D) H
2
O
2
content from butafenacil-treated rice seedlings. Seven-day-old rice seedlings were chal-
lenged with 0.1 µM butafenacil for 48 h. WT, wild type; FW, fresh weight. Different leers indicate
significant differences among the lines (Tukey’s HSD; p < 0.05).
4. Discussion
Melatonin acts as a master regulator in plant growth and development by orchestrat-
ing the expression of a diverse array of genes involved in primary and secondary metab-
olism [20,37]. SNAT plays roles in the penultimate and final steps of melatonin biosynthe-
sis, depending on the substrate [38].
A novel clade of archaeal SNAT showing no sequence identity to either animal
AANAT or plant SNAT genes was recently reported [20]. Thereafter, human Naa50 as a
functional orthologue of archaeal SNAT was confirmed to exhibit SNAT enzyme activity,
and its ectopic overexpression was functionally linked to melatonin biosynthesis in rice
[18]. Using human Naa50 as a query sequence, we screened for possible orthologues of
SNAT genes in the alveolate subgroup of the Stramenopila, Alveolata, and Rhizaria (SAR)
taxon. Alveolates comprise four major lineages: Chromerida, Apicomplexa, ciliates (Cili-
ophora), and dinoflagellates [1]. Many human Naa50 orthologues were discovered in the
genomes of alveolates with amino acid sequence identity ranging from 33% to 44%.
Among them, the ciliate S. lemnae SNAT (SlSNAT) showed 38% identity to human Naa50,
whereas the dinoflagellate Polarella glacialis SNAT had 36% identity. The predicted prod-
ucts of these SNAT orthologues showed high degrees of amino acid sequence identity
although the coenzyme-A-binding pocket sequences were poorly conserved (Figure 8).
On phylogenetic analysis, SlSNAT was closer to the dinoflagellate clade than the ciliate
(Ciliophora) clade. Melatonin was first identified in the dinoflagellate Lingulodinium poly-
edra (also named Gonyaulax polyedra) in 1989 [39] and was subsequently found in many
other dinoflagellate species, including Symbiodinium sp. [40,41]. Additionally, melatonin
was quantified in the ciliate Tetrahymena thermophila [40]. Although melatonin was de-
tected in these dinoflagellate and ciliate species, SNAT genes have not been cloned [1].
Figure 7. Enhanced tolerance of SlSNAT-overexpressing transgenic rice plants against peroxidiz-
ing herbicide butafenacil. (A) Photograph of rice seedlings after butafenacil treatment. (B) Effect
of butafenacil treatment on cellular leakage. (C) MDA production from butafenacil-treated rice
seedlings. (D) H
2
O
2
content from butafenacil-treated rice seedlings. Seven-day-old rice seedlings
were challenged with 0.1
µ
M butafenacil for 48 h. WT, wild type; FW, fresh weight. Different letters
indicate significant differences among the lines (Tukey’s HSD; p< 0.05).
Antioxidants 2024,13, 1177 10 of 13
4. Discussion
Melatonin acts as a master regulator in plant growth and development by orches-
trating the expression of a diverse array of genes involved in primary and secondary
metabolism [
20
,
37
]. SNAT plays roles in the penultimate and final steps of melatonin
biosynthesis, depending on the substrate [38].
A novel clade of archaeal SNAT showing no sequence identity to either animal AANAT
or plant SNAT genes was recently reported [
20
]. Thereafter, human Naa50 as a functional
orthologue of archaeal SNAT was confirmed to exhibit SNAT enzyme activity, and its
ectopic overexpression was functionally linked to melatonin biosynthesis in rice [
18
].
Using human Naa50 as a query sequence, we screened for possible orthologues of SNAT
genes in the alveolate subgroup of the Stramenopila, Alveolata, and Rhizaria (SAR) taxon.
Alveolates comprise four major lineages: Chromerida, Apicomplexa, ciliates (Ciliophora),
and dinoflagellates [
1
]. Many human Naa50 orthologues were discovered in the genomes
of alveolates with amino acid sequence identity ranging from 33% to 44%. Among them,
the ciliate S. lemnae SNAT (SlSNAT) showed 38% identity to human Naa50, whereas the
dinoflagellate Polarella glacialis SNAT had 36% identity. The predicted products of these
SNAT orthologues showed high degrees of amino acid sequence identity although the
coenzyme-A-binding pocket sequences were poorly conserved (Figure 8). On phylogenetic
analysis, SlSNAT was closer to the dinoflagellate clade than the ciliate (Ciliophora) clade.
Melatonin was first identified in the dinoflagellate Lingulodinium polyedra (also named
Gonyaulax polyedra) in 1989 [
39
] and was subsequently found in many other dinoflagellate
species, including Symbiodinium sp. [
40
,
41
]. Additionally, melatonin was quantified in the
ciliate Tetrahymena thermophila [
40
]. Although melatonin was detected in these dinoflagellate
and ciliate species, SNAT genes have not been cloned [1].
In the dinoflagellate L. polyedra, levels of both melatonin and 5-MT increased in re-
sponse to low temperatures, in conjunction with the circadian rhythmicity of melatonin
(showing a peak at night) [
42
,
43
]. Additionally, TPH, the first committed step enzyme
for melatonin biosynthesis in animals, exhibited a circadian rhythm with high amplitude
during the light period, antiphasic to the rhythm of melatonin [
44
]. The product of TPH en-
zyme catalysis is 5-hydroxytryptophan (5-HTP), which plays a key role in bioluminescence
in L. polyedra. Furthermore, the dinoflagellate genus Symbiodinium exhibited melatonin
rhythm with a nocturnal peak, although the diel pattern of melatonin levels did not persist
under constant dark conditions [
41
]. The dark-induced melatonin increase is believed to be
caused by the enhanced photoconsumption of melatonin by free radicals. Taken together,
these observations indicated that melatonin can regulate circadian rhythm, as in L. polyedra;
it also plays roles in antioxidant defense against free radicals generated from either cold
stress or photosynthesis in these unicellular photosynthesizing algae [45].
By analogy, there is evidence that ciliate T. thermophila also produces melatonin, indi-
cating that alveolates have a capacity to synthesize melatonin similar to the capacities of
animals, plants, and fungi [
10
]. There have been no previous studies of melatonin biosyn-
thesis in the ciliate S. lemnae, but the genome of S. lemnae reportedly carries an archaeal
SNAT orthologue, the predicted product of which exhibits 38% amino acid identity to
human Naa50. Purified recombinant SlSNAT protein has similar enzymatic characteristics
(optimal pH, temperature, and substrate preference) to the archaeal SNAT orthologue
protein products (Figures 3and 4), although there is some variation in kinetics among these
proteins [
18
,
20
,
21
]. As for the possible function of melatonin in S. lemnae, it is presumed
that melatonin may not only act as an antioxidant against various abiotic stresses but also
be involved in protein quality control during growth as shown in plants and animals [7].
Eukaryotic phototrophs comprise three taxa: Excavata, SAR, and Archaeplastida.
This is the first SNAT gene cloned in a eukaryotic phototroph outside the Archaeplastida.
Melatonin synthesis and SNAT genes have been reported and cloned from two of the three
Archaeplastida taxa: Rhodophyceae and Viridiplantae, but not Glaucophyta [
11
,
46
,
47
].
Melatonin was reported in the Excavata, such as Euglena gracilis, and in the SAR clade,
including dinoflagellates and ciliates [
1
]. Because the dinoflagellates are ecologically impor-
Antioxidants 2024,13, 1177 11 of 13
tant phytoplankton in marine environments and their genomes include SNAT orthologues
(Figure 8), further detailed molecular genetic analyses based on these SNAT genes will
provide new insights into the biological functions of melatonin in these organisms.
Antioxidants 2024, 13, x FOR PEER REVIEW 11 of 14
Figure 8. Sequence comparison and phylogenetic tree of SNAT in the Ciliophora and dinoflagellates.
(A) Consensus amino acid sequences among three SNAT proteins including the human Naa50, the
ciliate Stylonichia lemnae SNAT, and the dinoflagellate Polarella glacialis SNAT. Bold red leers indi-
cate consensus amino acids. Dashes denote gaps for maximizing alignment of conserved residues.
A coenzyme-A-binding pocket is underlined. (B) Phylogenetic tree analysis of SNAT proteins from
the ciliates and dinoflagellates. GenBank accession numbers of various SNAT genes are as follows:
human Naa50 (BAB14397), Cladocopium goreaui (CAI3999280); Effrenium voratum (CAJ1361560); Po-
larella glacialis (CAK0876941); Stylonichia lemna (CCKQ01002460); Paramecium sonneborni
(CAD8056267); Pseudocohnilembus persalius (KRX00195); Tetrahymena thermophila SB210
(XP_001025216); Ichthyophthirius multifiliis (XP-004035125). The scale bar represents 0.3 substitutions
per site.
In the dinoflagellate L. polyedra, levels of both melatonin and 5-MT increased in re-
sponse to low temperatures, in conjunction with the circadian rhythmicity of melatonin
(showing a peak at night) [42,43]. Additionally, TPH, the first commied step enzyme for
melatonin biosynthesis in animals, exhibited a circadian rhythm with high amplitude dur-
ing the light period, antiphasic to the rhythm of melatonin [44]. The product of TPH en-
zyme catalysis is 5-hydroxytryptophan (5-HTP), which plays a key role in biolumines-
cence in L. polyedra. Furthermore, the dinoflagellate genus Symbiodinium exhibited mela-
tonin rhythm with a nocturnal peak, although the diel paern of melatonin levels did not
persist under constant dark conditions [41]. The dark-induced melatonin increase is be-
lieved to be caused by the enhanced photoconsumption of melatonin by free radicals.
Taken together, these observations indicated that melatonin can regulate circadian
rhythm, as in L. polyedra; it also plays roles in antioxidant defense against free radicals
generated from either cold stress or photosynthesis in these unicellular photosynthesizing
algae [45].
By analogy, there is evidence that ciliate T. thermophila also produces melatonin, in-
dicating that alveolates have a capacity to synthesize melatonin similar to the capacities
of animals, plants, and fungi [10]. There have been no previous studies of melatonin bio-
synthesis in the ciliate S. lemnae, but the genome of S. lemnae reportedly carries an archaeal
SNAT orthologue, the predicted product of which exhibits 38% amino acid identity to hu-
man Naa50. Purified recombinant SlSNAT protein has similar enzymatic characteristics
(optimal pH, temperature, and substrate preference) to the archaeal SNAT orthologue
protein products (Figures 3 and 4), although there is some variation in kinetics among
Figure 8. Sequence comparison and phylogenetic tree of SNAT in the Ciliophora and dinoflagellates.
(A) Consensus amino acid sequences among three SNAT proteins including the human Naa50, the
ciliate Stylonichia lemnae SNAT, and the dinoflagellate Polarella glacialis SNAT. Bold red letters indicate
consensus amino acids. Dashes denote gaps for maximizing alignment of conserved residues. A
coenzyme-A-binding pocket is underlined. (B) Phylogenetic tree analysis of SNAT proteins from the
ciliates and dinoflagellates. GenBank accession numbers of various SNAT genes are as follows: hu-
man Naa50 (BAB14397), Cladocopium goreaui (CAI3999280); Effrenium voratum (CAJ1361560); Polarella
glacialis (CAK0876941); Stylonichia lemna (CCKQ01002460); Paramecium sonneborni (CAD8056267);
Pseudocohnilembus persalius (KRX00195); Tetrahymena thermophila SB210 (XP_001025216); Ichthyophthir-
ius multifiliis (XP-004035125). The scale bar represents 0.3 substitutions per site.
5. Conclusions
A novel archaeal SNAT clade was identified showing no apparent sequence identity to
either animal AANAT or plant SNAT. Archaeal SNAT orthologues have recently been cloned
from human [
18
], E. coli [
21
], and rice [
28
]. In this study, an orthologue of archaeal SNAT
from S. lemnae was cloned, and its product was characterized. SlSNAT overexpression in
the rice genome increased melatonin content relative to wild type. SlSNAT-OE rice plants
exhibited increased tolerance to treatment with the peroxidizing herbicide butafenacil,
as indicated by the lower levels of MDA and H
2
O
2
compared with wild-type controls,
indicating that as a potent antioxidant melatonin plays a role in defense against oxidative
stress in rice by lowering ROS levels. However, we cannot rule out that other roles of
melatonin, such as the induction of many antioxidant enzymes and protein quality control
proteins, may also contribute to the beneficial effects of melatonin on oxidative stress.
Author Contributions: Conceptualization, K.B.; data curation, K.L. and K.B.; formal analysis, K.L.
and K.B.; writing—original draft, K.B.; and writing—review and editing, K.B. All authors have read
and agreed to the published version of the manuscript.
Funding: This research was supported by grants by the Basic Science Research Program of the Na-
tional Research Foundation of Korea (NRF-2021R1I1A2042237) funded by the Ministry of Education.
Antioxidants 2024,13, 1177 12 of 13
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Data presented in this study are available within the article.
Conflicts of Interest: The authors declared no conflicts of interest.
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