Knockout of arabidopsis serotonin N-acetyltransferase-2 reduces melatonin levels and delays flowering

Article abstract of DOI:10.3390/biom9110712

Melatonin plays roles in both plant growth and defense. Serotonin N-acetyltransferase (SNAT) catalyzes formation of N-acetylserotonin (NAS) from serotonin. Plants contain two SNAT isogenes, which exhibit low-level amino acid homology. We studied the Arabidopsis thaliana SNAT2 (AtSNAT2) gene; we prepared recombinant SNAT2 protein and characterized a snat2 knockout mutant. The SNAT2 protein exhibited 27% amino acid homology with SNAT1; the K_m was 232 μM and the V_max was 2160 pmol/min/mg protein. Melatonin inhibited SNAT enzyme activity in vitro. SNAT2 mRNA was abundantly expressed in flowers; the melatonin content of flowers of the snat2 mutant was significantly less than that of wild-type flowers. The mutant exhibited delayed flowering and reductions in leaf area and biomass compared to the wild type. Delayed flowering was attributable to reductions in the expression levels of the gibberellin biosynthetic genes ent-kaurene synthase (KS) and FLOWERING LOCUS T (FT).

Full text of DOI:10.3390/biom9110712

biomolecules
Article
Knockout of Arabidopsis Serotonin
N-Acetyltransferase-2 Reduces Melatonin Levels
and Delays Flowering
Hyoung Yool Lee †, Kyungjin Lee † and Kyoungwhan Back *
Division of Food Technology and Biotechnology, College of Agriculture and Life Sciences, Chonnam National
University, Gwangju 61186, Korea; xanthine@naver.com (H.Y.L.); nicekj7@hanmail.net (K.L.)
* Correspondence: kback@chonnam.ac.kr; Tel.: +82-62-530-2165
† These authors contributed equally to the article.
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 17 October 2019; Accepted: 4 November 2019; Published: 6 November 2019
Abstract: Melatonin plays roles in both plant growth and defense. Serotonin N-acetyltransferase
(SNAT) catalyzes formation of N-acetylserotonin (NAS) from serotonin. Plants contain two SNAT
isogenes, which exhibit low-level amino acid homology. We studied the Arabidopsis thaliana SNAT2
(AtSNAT2) gene; we prepared recombinant SNAT2 protein and characterized a snat2 knockout mutant.
The SNAT2 protein exhibited 27% amino acid homology with SNAT1; the Km was 232 µM and the
Vmax was 2160 pmol/min/mg protein. Melatonin inhibited SNAT enzyme activity in vitro. SNAT2
mRNA was abundantly expressed in flowers; the melatonin content of flowers of the snat2 mutant
was significantly less than that of wild-type flowers. The mutant exhibited delayed flowering and
reductions in leaf area and biomass compared to the wild type. Delayed flowering was attributable to
reductions in the expression levels of the gibberellin biosynthetic genes ent-kaurene synthase (KS) and
FLOWERING LOCUS T (FT).
Keywords: Arabidopsis; recombinant SNAT2; flowering; melatonin; snat2 knockout
1. Introduction
The recent identification of an Arabidopsis phytomelatonin receptor implied that melatonin may
serve as a plant hormone [
leaf senescence [ ], quality control of the endoplasmic reticulum [
metabolite synthesis [ ], somatic embryogenesis [ ], stomatal closure [
and leaf development [11]. Melatonin plays roles in plant responses to both biotic and abiotic
stressors [12 14], mediating tolerance to drought [15], high salt levels [16], high temperatures [17], cold
1,
2
]. Melatonin plays many biological roles in plants, being involved in
], plant growth [ ], secondary
], skotomorphogenesis [10],
3
4
5,6
7,8
9
1
–
conditions [18], heavy metals [19–22], and pathogens [23,24].
All plant melatonin biosynthetic genes have been cloned [25]. The genes encoding the last
two steps in melatonin biosynthesis are serotonin N-acetyltransferase (SNAT) and N-acetylserotonin
O-methyltransferase (ASMT). Plants contain two SNAT isogenes [25]; the rice genome contains three
copies of ASMT [26]. SNAT plays important roles in both melatonin function and biosynthesis.
Suppression of rice SNAT2 decreased levels of brassinosteroids (plant hormones) and induced a dwarf
phenotype, but, suppression of rice SNAT1 did not result in such effects, although melatonin levels
were similarly decreased [10]. The in vitro enzyme kinetics of rice SNAT1 and SNAT2 differed [25].
The SNAT1 genes of various plants (and cyanobacteria) have been cloned and characterized [27
–29],
but SNAT2 has been cloned only from rice [30] and grapevine [31].
Although many physiological functions played by melatonin have been reported, there has not
been any molecular genetic study on the effect of flowering. Plant flowering is initiated by physical
Biomolecules 2019, 9, 712; doi:10.3390/biom9110712
www.mdpi.com/journal/biomolecules

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factors such as the photoperiod and vernalization, and by gibberellic acid (GA; a hormone; [32]).
The key photoperiod-regulated flowering gene is the night-labile CONSTANT (CO), a zinc finger protein
that activates FLOWERING LOCUS T (FT), which in turn promotes floral induction. Vernalization
accelerates flowering by decreasing the expression of FLOWERING LOCUS C (FLC), which encodes a
MADS-box transcription factor that represses FT. In addition to the above physical factors, GA also
directly or indirectly controls FT expression.
Here, we cloned Arabidopsis thaliana SNAT2 and explored the kinetics of the recombinant protein.
We investigated an SNAT2 knockout mutant (snat2) in which decreased melatonin levels delayed
flowering and retarded growth.
2. Materials and Methods
2.1. Plant Materials and Growth
Arabidopsis thaliana wild-type Col-0 and the knockout snat2 mutant line (SALK_062388) were
grown at 22 ◦C under 60% relative humidity, a 14 h photoperiod, and a photon flux density of 50
µ
mol
m−2
s
−1. The Arabidopsis snat2 line (with a T-DNA insert in SALK_062388) was obtained from the
Arabidopsis Biological Resource Center (Ohio State University, Columbus, OH, USA). The line was
genotyped using the T-DNA express: Arabidopsis gene mapping tool (http://signal.salk.edu/). Tobacco
plants (Nicotiana benthamiana) were grown under a 16 h light (28 ◦C)/8 h dark (22 ◦C) photoperiod
featuring white light (100 µ
mol photons m−2 s−1). Flowering times were defined by the days to bolting
and the numbers of rosette leaves at bolting (n = 20). Leaf area was measured using Fuji ImageJ
software [33]. Rosette leaves were photographed adjacent to a ruler; pixels were then converted to
metric units. All data were processed with the aid of Microsoft Excel 2010. All experiments were
performed in triplicate and were repeated at least three times.
2.2. Homology and Phylogenetic Analysis of AtSNAT2
Amino acid sequence homology was explored with the aid of the BLASTp tool; we interrogated
the nonredundant, protein sequence databases of the National Center for Biotechnology Information
(http://www.ncbi.nlm.nih.gov/). BLAST-Explorer was used to generate sequence alignments and
phylogenetic trees [34].
2.3. Cloning of AtSNAT2 and Purification of Recombinant AtSNAT2 Protein from Escherichia Coli
Total RNA of 3-week-old Arabidopsis leaves was isolated using a NucleoSpin RNA plant kit
(Macherey-Nagel, Duren, Germany) and 1
transcriptase (Thermo Scientific Fermentas, St. Leon-Ro, Germany) after addition of 500 ng of
oligo(dT)18 primer, at 42 ◦C for 1 h. The cDNA obtained (0.2
L) served as the template for PCR-based
µg thereof was reverse-transcribed using RevertAid
µ
cloning. Reverse transcription (RT)-PCR was employed to clone full-length AtSNAT2 (GenBank
accession number BT005218) using the primer pair 50-AAA AAG CAG GCT CCA TGT TTC TCG GAG
GCA-30 and 50-AGA AAG CTG GGT TTA TTT CTT GTT TCT CTG-30. The initial AtSNAT2 PCR product
was cloned into the T&A vector (RBC Bioscience, New Taipei City, Taiwan) and the integrity thereof
confirmed via sequencing (Bioneer, Daejeon, Korea). Prior to AtSNAT2 expression and purification,
the initial RT-PCR product was amplified using a primer pair containing attB recombination sequences
(forward primer, 50-GGG GAC AAG TTT GTA CAA AAA AGC AGG CT-30; reverse primer, 50-GGG
GAC CAC TTT GTA CAA GAA AGC TGG GT-30). The resulting AtSNAT2 PCR product was cloned
via the Gateway recombination reaction into the pDONR221 vector (Invitrogen, Carlsbad, CA, USA),
and then into the destination vector pET300/NT-DEST (Invitrogen). The pET300-AtSNAT2 plasmid
was transformed into E. coli strain BL21(DE3) (Invitrogen). An aliquot (10 mL) of an overnight culture
grown in the presence of ampicillin (50 mg/L) was inoculated into 100 mL of Terrific Broth [20 g/L
Bacto-tryptone, 24 g/L Bacto-yeast extract, glycerol 4 mL/L, and phosphate buffer (0.017 M KH2PO4
and 0.072 M K2HPO4)] containing 50 mg/L ampicillin and incubated at 37 °C until the optical density

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at 600 nm attained 1.0. After addition of isopropyl-
β-D-thiogalactopyranoside (IPTG; Sigma, St. Louis,
MO, USA) to 1 mM, the culture was grown at 28 °C with shaking at 180 rpm for 5 h. The protein was
purified via affinity (Ni2+) chromatography according to the recommendations of the kit manufacturer
(Qiagen, Tokyo, Japan).
2.4. AtSNAT Enzyme Kinetics
Purified recombinant AtSNAT2 protein was incubated in a final volume of 100 µL with 0.5 mM
serotonin or 5-methoxytryptamine (5-MT), and 0.5 mM acetyl-CoA, in 100 mM potassium phosphate
(at pH 7.8 or other pH values) at 45 ◦C (or other temperatures) for 1 h. Reaction products including
N-acetylserotonin (NAS) and melatonin were identified with the aid of high-performance liquid
chromatography (HPLC). In brief, each reaction was terminated by addition of 20
30 L methanol and 10 L aliquots were subjected to reverse-phase HPLC (Waters, Milford, MA, USA)
on a SunFire C18 column (Waters; 4.6 150 mm) eluted using an isocratic profile of 6% (v/v) methanol
µL acetic acid and
µ
µ
×
for NAS or 10% (v/v) methanol (both in water with 0.3% [v/v] trifluoroacetic acid) at a flow rate of
1 mL/min. Compounds were detected via absorbance at 280 nm. The substrate affinity (Km) and
maximum reaction rate (Vmax) were calculated using Lineweaver–Burk plots. Protein concentrations
were determined using the Bradford method employing a protein-specific dye (Bio-Rad, Hercules, CA,
USA). All analyses were performed in triplicate.
2.5. Subcellular Localization of AtSNAT2
We used the pER-mCherry vector [the kind gift of Dr. H.G. Kang (Texas State University,
San Marcos, TX, USA)] to localize the AtSNAT2 protein. Full-length AtSNAT2 was PCR-amplified
using a primer set containing AscI sites (GGCGCGCC). The resulting product was cloned into
the T&A vector (RBC Bioscience) and the AtSNAT2 insert was digested with AscI and cloned into
the AscI site of the binary vector, pER8-mCherry, containing the estrogen-inducible XVE promoter.
The pER8-AtSNAT2-mCherry plasmid was transferred into Agrobacterium tumefaciens strain GV2260
using the freeze–thaw method. A confocal microscope (TCS-SP5; Leica, Wetzlar, Germany) was used
to evaluate transient within-cell protein expression, as described previously [35].
2.6. Total RNA Isolation and Reverse Transcription/Polymerase Chain Reaction
Total plant RNA was isolated using a NucleoSpin RNA plant kit (Macherey-Nagel, Duren,
Germany). Semi-quantitative reverse transcription (RT)-PCR analyses were performed as described
previously [21]. The primer sequences used for PCR amplification were as follows: SNAT1 (forward,
50-GCC AAG GAG ACC GTT AGT GA-30, reverse, 50-CTT TGG GTA CCA AAA CAT GCC-30); SNAT2
(forward, 50-ATG TTT CTC GGA GGC ACA AT-30, reverse, 50-ATT TCT TGT TTC TCT GTT TGC
GT-30); CCA1 (forward, 50-GTT GCA GCT GCT AGT GCT TG-30, reverse 50-CAA AGG CCT CAA
AAG AAA AAG A-30); TOC1 (forward, 50-TCT CCG GTG ACT TTT GTT GA-30, reverse 50-TGG CCA
AAT CAG AAC TAG GG-30); KS (forward, 50-CCA AGT TGA TCT GGC AGG TA-30, reverse 50- TTG
TCT CCT AAA ATC AAT TTT CCT C-30); MYB33 (forward, 50-TTG TTC TTG GAG CAA CA TGC-30,
reverse 50-TGC TTG GCA GTT GCT AGT C-30); FT (forward, 50-GGT GGA GAA GAC CTC AGG
AA-30, reverse 50-CTC ATT TTC CTC CCC CTC TC-30); BIP2 (forward, 50-GCA GGA GGA GAA TCA
TCG AC-30, reverse 50-AAA GAG AAC GTC CAG GGA GA-30); and PR1 (forward, 50-CTA TAT AAG
GAA GTT CAT TT-30, reverse 50-CTA TAT AAG GAA GTT CAT TT-30). EF-1a transcript levels served
to normalize all signals (forward, 50-TGG TGA CGC TGG TAT GGT TA-30, reverse 50- CAT CAT TTG
GCA CCC TTC TT-30).
2.7. Melatonin Quantification
Arabidopsis leaves (0.2 g) were ground in liquid nitrogen and thoroughly homogenized in
2 mL chloroform. After centrifugation at 12,000
evaporated to dryness, and the residues were dissolved in 1 mL amounts of methanol; 10
×
g for 10 min, the supernatants were decanted and
L aliquots
µ

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were used for melatonin quantitation via liquid chromatography/dual mass spectrometry (LC-MS/MS;
Shimadzu, Kyoto, Japan) as described previously [36]. A mass transition from m/z 233.1 to m/z 174.0
identified melatonin; the retention time was 3.65 min. AB Sciex Analyst software (version 1.5.2) was
used for data integration.
2.8. Data Analysis
We used the Student’s t-test of SigmaPlot ver. 10 (Systat Software, Point Richmond, CA, USA).
p < 0.05 was considered to indicate statistical significance.
3. Results
3.1. Cloning and Phylogeny of AtSNAT2
SNAT is the penultimate enzyme of the melatonin biosynthetic pathway; plants contain two
SNAT isogenes [30]. Plant SNAT1 genes have been relatively well studied [25], but the SNAT2 genes
of only rice [10] and grapevine [31] have been evaluated. No plant SNAT2 knockout mutant has yet
been investigated. Here, we cloned the rice SNAT2 homolog of Arabidopsis (AtSNAT2). The amino
acid sequences of the rice SNAT2 and AtSNAT2 were 48% identical (Figure 1A). Plant SNAT1 and
SNAT2 share an amino acid identity level <27%. The two AtSNAT isogenes belong to different clades,
implying that they arose via independent evolution rather than gene duplication (Figure 1B).
Figure 1. Amino acid comparisons among, and the phylogenetic trees of, SNAT isogenes. (
amino acid sequences of OsSNAT2 and AtSNAT2. ( ) Phylogenetic analyses of rice and Arabidopsis
SNAT isogenes. Identity is denoted by stars. Gaps are noted as dashes. Scale bar: substitution value of
A) Predicted
B
0.2/site. Phylogenetic analysis was performed with the aid of BLAST-Explorer (www.phylogeny.fr)
The GenBank accession numbers are OsSNAT1 (AK059369), OsSNAT2 (AK068156), AtSNAT1
(At1g32070), and AtSNAT2 (At1g26220; BT005218).
.
3.2. Subcellular Location of AtSNAT2
To evaluate whether AtSNAT2 is also expressed in chloroplasts, we transiently expressed the
AtSNAT2-mCherry fusion protein in tobacco leaves. Figure 2 shows that the protein localized to
chloroplasts; the chlorophyll and mCherry fluorescences overlapped precisely. Thus, AtSNAT2 is
expressed in chloroplasts, as are other plant SNAT proteins [30,31,35].

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Figure 2. Subcellular localization of AtSNAT2. (A) Red fluorescence of AtSNAT2-mCherry. (B) Cyan
fluorescence of chlorophyll. ( ) Merged fluorescent images (A+B). Tobacco leaves were infiltrated with
C
Agrobacterium tumefaciens GV2260 expressing the XVG-inducible AtSNAT2-mCherry fusion protein.
Bars: 5 µm.
3.3. AtSNAT2 Enzyme Kinetics
We expressed two AtSNAT2 gene constructs in E. coli; the chloroplast-targeting sequence inhibits
heterologous protein expression in that bacterium [37]. We thus expressed both full-length AtSNAT2
and an N-terminal-37-amino acid-truncated protein. TargetP analysis predicted the presence of an
N-terminal 37-amino acid transit sequence [38]. We purified both recombinant proteins via Ni2+
affinity chromatography; both featured N-terminal hexa-histidine fusions (Figure 3). IPTG enhanced
the expression levels of both proteins, but the extent of inducibility and the expression levels of the
N-terminal-truncated ∆37AtSNAT2 were greater than those of the full-length protein.
Figure 3. Affinity purification of AtSNAT2 from Escherichia coli. (
AtSNAT2 protein. ( ) Purification of the protein lacking the N-terminal 37 amino acids. E. coli
BL21 (DE3) cells harboring pET300-AtSNAT2 and pET300- 37AtSNAT2 were employed for protein
purification. M, molecular mass standards; lane 1, total proteins of 15 L aliquots of bacterial cultures
grown in the absence of IPTG; lane 2, total proteins of 15 L aliquots of bacterial cultures grown in
the presence of IPTG; lane 3, 20 g of soluble proteins; lane 4, 10 g of protein purified via affinity
A) Purification of the complete
B
∆
µ
µ
µ
µ
chromatography. Protein samples were separated on 12% (w/v) SDS-PAGE gels and stained with
Coomassie blue.
The pH and temperature optima in terms of enzymic activity were determined. Figure 4 shows
that the total SNAT activity of the full-length protein was greater than that of the truncated protein
under all conditions tested. The optimal pH values in terms of activity were pH 7.8 for both proteins,
followed by pH 8.8 (Figure 4A). SNAT enzyme activity was 10-fold lower in the pH range pH 5.4–6.5
than at pH 7.8. Optimal SNAT enzyme activity at relatively high pH values is characteristic of other
plant SNAT2 enzymes [30
but only 45 °C for 37AtSNAT2. Surprisingly, AtSNAT2 retained 30% of its peak activity even at 95 °C
37AtSNAT2 exhibited no activity at that temperature (Figure 4B). Neither the truncated rice nor
grape enzyme exhibited any activity at 95 °C 30 31]. In contrast, rice 83SNAT1 exhibited enzyme
activity comparable to that of AtSNAT2 at 95 °C 35], implying that plant SNAT enzymes are more
thermostable than other melatonin biosynthetic enzymes such as ASMT [35].
,31]. The optimal temperatures for activity were 45 °C and 55 °C for AtSNAT2,
∆
;
∆
[
,
∆
[

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Figure 4. The enzymic activity of recombinant AtSNAT2. (
(B) The activity at various temperatures. Enzyme activity was measured by assaying N-acetylserotonin
(NAS) production by 0.5 g recombinant protein from 0.5 mM serotonin for 1 h under various conditions.
A) The activity at various pH values.
µ
In assays performed at the optimal pH and temperature, we next determined Km and Vmax values
(Figure 5). The Km values for NAS and 5-MT were 232 and 630 M, respectively. The Vmax values
µ
for NAS and 5-MT were 2160 and 3360 pmol/min/mg protein, respectively. The catalytic efficiency
(Vmax/Km) for NAS was 9.3 and that for 5-MT was 5.3, indicating that AtSNAT favors NAS synthesis
from serotonin rather than melatonin synthesis from 5-MT. The effects of 5-MT on enzyme activity
were assessed by measuring the conversion of serotonin to NAS in the presence of 5-MT. Figure 6
shows that NAS production (from 500
and further fell fourfold when 100 M of 5-MT was added. The decrease in NAS production was
not proportional to the 5-MT concentration. Notably, the NAS and melatonin production levels were
similar when the substrate levels were 500 M, implying that the affinity of SNAT for serotonin and
5-MT is similar when substrate levels are high.
µM serotonin) was inhibited by the addition of 50 µM 5-MT,
µ
µ
Figure 5. The Michaelis–Menten kinetics of recombinant AtSNAT2. (
serotonin as substrate. ( ) The Km and Vmax values with 5-methoxytryptamine (5-MT) as substrate. Km and
max values were determined using Lineweaver–Burk plots. The enzymatic products (N-acetylserotonin
and melatonin) were assayed using high-performance liquid chromatography (HPLC).
A) The Km and Vmax values with
B
V
Figure 6. The effect of 5-MT on SNAT enzyme activity. (
) Inhibition of NAS synthesis by 5-MT. ( ) Melatonin production in the presence of serotonin (0.5 mM)
and varying levels of 5-MT. All assays were performed in the presence of 0.5 g AtSNAT2, 0.5 mM
serotonin, and various levels of 5-MT. The data are the means of three replicates.
A) A schematic of the SNAT enzymic reaction.
(B
C
µ

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We explored whether AtSNAT1 [28] and AtSNAT2 activity was feedback-regulated by melatonin.
Recombinant AtSNAT2 activity was inhibited by addition of 1 M melatonin to an extent twofold
µ
that of the control (Figure 7), but AtSNAT1 activity was not affected by any tested concentration of
melatonin, implying that the modes of action of the SNAT isoenzymes differ.
Figure 7. Effect of melatonin on SNAT enzyme activity. (
) Melatonin-mediated inhibition of NAS synthesis by recombinant AtSNAT1. (
inhibition of NAS synthesis by recombinant AtSNAT2. AtSNAT1 was prepared as described
previously [28]. The SNAT enzyme assay was performed in the presence of 0.5 g AtSNAT2, 0.5 mM
A
) A schematic of the enzymic reaction.
(B
C
) Melatonin-mediated
µ
serotonin, and various levels of melatonin. The data are the means of three replicates. Asterisks (*)
denote significant differences compared to the Col-0 controls (Student’s t-test; p < 0.05).
3.4. AtSNAT2 Expression and the Phenotype of the snat2 Knockout Arabidopsis Mutant
To explore directly whether AtSNAT2 engages in melatonin synthesis and the physiological role
played by melatonin, we investigated a T–DNA insertional snat2 knockout mutant (SALK_062388)
(Figure 8A). We first measured SNAT1 and SNAT2 transcript levels in various tissues via RT-PCR
analysis. SNAT1 mRNA levels were highest in mature leaves; SNAT2 mRNA was abundant in flowers
and young leaves; the two isogenes thus exhibited different expression patterns (Figure 8B). RT-PCR of
material from one-week-old leaves showed that SNAT2 mRNA was absent in the mutant (Figure 8C).
Melatonin levels were 50% lower in flowers of the snat2 mutant than in those of the wild-type Col-0,
but the melatonin levels of leaves did not differ, implying that SNAT2 expression is tissue-specific
in terms of both mRNA and melatonin synthesis (Figure 8D). In line with this implication, the snat2
mutant exhibited delayed flowering, as evidenced by increased leaf numbers (Figure 9A–C). During
bolting, the height of the snat2 mutant was less than that of the wild type, but the mutant attained the
wild-type height eventually (Figure 9D). The leaf area of the snat2 mutant was less than that of the
wild type, reducing the biomass of the mutant (Figure 9E,F).

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Figure 8. A schematic of Arabidopsis snat2 knockout and SNAT2 mRNA expression. (
structure of the Arabidopsis SNAT2 gene (At1g26220). Exons and introns are denoted by solid and open
boxes, respectively. The location of the T-DNA insertion is arrowed (not to scale). ( ) Tissue-specific
expression levels of AtSNAT1 and AtSNAT2 as revealed by RT-PCR. ( ) RT-PCR analysis of SNAT2
in wild-type Col-0 and snat2 mutant. ( ) Melatonin levels in leaves and flowers. The numbers in
parentheses are the PCR cycle numbers. EF-1a served as a loading control, as described previously [ ].
A) Genomic
B
C
D
4
An asterisk (*) indicates a significant difference compared to the wild-type Col-0 (Student’s t-test;
p < 0.05).
Figure 9. Delayed flowering of the Arabidopsis snat2 knockout mutant. (
) The rosette leaf phenotype. ( ) The rosette leaf numbers of the wild-type Col-0 and the snat2
mutant. ( ) Stem lengths over time and a comparison of rosette leaf areas. ( ) Leaf lengths over time.
) Biomass values (n = 10). Stem lengths were measured after the wild type commenced bolting
A) Phenotypes at flowering.
(
B
C
D
E
(
F
(about 34 d after germination). Bars: 1 cm. Asterisks (*) denote significant differences compared to the
wild-type Col-0 (Student’s t-test; p < 0.05).
To define the flowering pathway affected by the snat2 mutation, we used RT-PCR to measure
gene expression levels. As shown in Figure 10, the expression levels of photoperiod pathway-related
flowering genes, including Circadian Clock Associated 1 (CCA1) and Timing of CAB expression 1 (TOC1),
were similar in the mutant and wild type over the 24 h day/night cycle (Figure 10A). However, a key
gene involved in GA biosynthesis, ent-kaurene synthase (KS), was significantly suppressed, whereas
GA-responsive negative transcription factor MYB33 was enhanced in the mutant compared to the
wild type, implying that changes in the GA-related pathway (and not the photoperiod pathway)
explained the delayed flowering of the snat2 mutant. Reductions in GA synthesis and enhanced
MYB33 expression decreased levels of FT florigen, delaying flowering (Figure 10C). We explored

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whether flowering-related gene expression was affected by exogenous melatonin (1
µM) that infiltrated
leaves over 2 h. As shown in Figure 10B, the expression levels of melatonin-induced representative
marker genes such as BIP2 and PR1 increased markedly [ ], but melatonin suppressed expression of
4
the KS, MYB33, and FT genes, consistent with a previous finding that exogenous melatonin delays
flowering [39]. KS, MYB33, and FT suppression by exogenous melatonin was in sharp contrast with
such suppression in the snat2 mutant, indicating that exogenous melatonin did not mimic the effects of
endogenous melatonin on flowering.
Figure 10. The daily rhythms of flowering-related gene expression. (
leaves as revealed by RT-PCR. ( ) Gene expression levels after addition of exogenous melatonin.
(C) A model of delayed flowering in the snat2 mutant. EF-1a served as the loading control. Fully
A) Daily rhythms of 4-week-old
B
mature leaves of 4-week-old wild-type Col-0 Arabidopsis were infiltrated with 1 µM melatonin and
harvested 3 h later. Water served as the control.
4. Discussion
Given the many available Arabidopsis T-DNA knockout mutants, it is simple to explore the roles
played by genes of interest [40], including those involved in melatonin metabolism. Melatonin is
biosynthesized from tryptophan in four steps catalyzed by tryptophan decarboxylase (TDC), tryptamine
5-hydroxylase (T5H), SNAT, and ASMT [25]. The SNAT-catalyzed reaction is the penultimate step;
the SNAT gene family plays pivotal roles in terms of melatonin synthesis regulation in both plants and
animals [41]. Rice SNAT1 was first cloned in 2013 [37], followed by SNAT2 [30]; orthologs exist in all
plant taxa explored [27
in melatonin synthesis. It is known that all plant SNAT proteins explored to date were located in
chloroplasts [27 30 31 35]. However, single transgenic rice plants differ in terms of phenotype and stress
,30]. Both SNAT1 and SNAT2 are nucleus-encoded chloroplast proteins involved
,
,
,
response. Rice plants overexpressing SNAT1 are resistant to abiotic stressors such as senescence [21],
while SNAT1 suppression increased the susceptibility of rice plants to cold [42]. However, SNAT2
suppression conferred tolerance to several abiotic stressors, including both cold and heat [43], and was
associated with a novel dwarf-like phenotype combined with erect leaves [10]. Thus, the two SNAT
isogenes mediate different physiological responses in terms of melatonin synthesis in plants.
Recently, the grape SNAT2 gene was cloned and characterized [31]. The grapevine SNAT2 is 55%
identical (in terms of their amino acid sequences) to rice SNAT2. The optimal pH and temperature for
grape and rice SNAT2 activity are similar, but the catalytic efficiency (Vmax/Km) of the grape enzyme
is less than that of the rice enzyme [30]. Transgenic Arabidopsis overexpressing grape SNAT2 exhibit
enhanced melatonin synthesis and increased resistance to powdery mildew (compared to the wild
type) [31]. However, no knockout mutant of grape SNAT2 has yet been reported. Our Arabidopsis
SNAT2 knockout mutant (snat2) clearly exhibited delayed flowering (Figure 9), as confirmed via
molecular genetic analysis. Thus, melatonin affects plant flowering to some extent, but does not delay
flowering markedly.
In contrast to the effects of the snat2 mutation, exogenous melatonin retarded Arabidopsis flowering
by elevating the level of DELLA protein, the master regulator of GA production, in turn suppressing

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the expression of flowering genes, including SUPPRESSOR OF OVEREXPRESSION OF CONSTANT 1
(SOC1) and FLOWERING LOCUS T (FT) [39]. These contradictory results are explained by the fact that
high-dose melatonin (0.5 mM) retards the growth of Arabidopsis seedlings. The melatonin biosynthetic
mutant snat1 also exhibited delayed flowering (data not shown). We are currently studying the snat1
mutant in detail.
We found, for the first time, that SNAT2 was involved in melatonin synthesis in Arabidopsis and
was abundantly expressed in flowers. The snat2 mutant produced less melatonin than did the wild type,
in turn affecting GA biosynthesis and FT expression, both of which regulate flowering. We suggest that
the reduced melatonin levels are associated with less GA synthesis, and compromise FT expression,
in turn inhibiting flowering. The mechanism by which melatonin regulates GA synthesis remains to
be investigated.
5. Conclusions
Melatonin plays roles in both plant growth and defense. Serotonin N-acetyltransferase (SNAT)
catalyzes the formation of N-acetylserotonin (NAS) from serotonin; NAS is then converted to melatonin.
Plants contain two SNAT isogenes, which exhibit low-level amino acid homology. SNAT is the
penultimate enzyme of melatonin biosynthesis. This is the first study to characterize a snat2 mutant
genetically. The mutant exhibited delayed flowering; this is the first genetic indication that melatonin
is involved in flowering.
Author Contributions: Conceptualization, K.B.; data curation, H.Y.L. and K.L.; formal analysis, H.Y.L. and K.L.;
funding acquisition, K.B.; writing—original draft, K.B.; and writing—review and editing, K.B.
Funding: This research was supported by grants from the Research Fellow Program (NRF-2017R1A6A3A11028333)
and the Basic Science Research Program of the National Research Foundation of Korea (2017R1A2A2A05069253)
funded by the Ministry of Education, Republic of Korea.
Conflicts of Interest: The authors declare no conflict of interest.
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