Melatonin in Arabidopsis thaliana acts as plant growth regulator at low concentrations and preserves seed viability at high concentrations

Abstract

Since the discovery of melatonin in plants, several roles have been described for different species, organs, and developmental stages. Arabidopsis thaliana, being a model plant species, is adequate to contribute to the elucidation of the role of melatonin in plants. In this work, melatonin was monitored daily by UHPLC-MS/MS in leaves, in order to study its diurnal accumulation as well as the effects of natural and artificial light treatments on its concentration. Furthermore, the effects of exogenous application of melatonin to assess its role in seed viability after heat stress and as a regulator of growth and development of vegetative tissues were evaluated. Our results indicate that melatonin contents in Arabidopsis were higher in plants growing under natural radiation when compared to those growing under artificial conditions, and its levels were not diurnally-regulated. Exogenous melatonin applications prolonged seed viability after heat stress conditions. In addition, melatonin applications retarded leaf senescence. Its effects as growth promoter were dose and tissue-dependent; stimulating root growth at low concentrations and decreasing leaf area at high doses.
Copyright © 2015 Elsevier Masson SAS. All rights reserved.

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Research article
Melatonin in Arabidopsis thaliana acts as plant growth regulator at low
concentrations and preserves seed viability at high concentrations
Isma
el Gatica Hern
andez
a
, Federico Jos
e Vicente Gomez
a
, Soledad Cerutti
b
,
María Ver
onica Arana
c
, María Fernanda Silva
a
,
*
a
Instituto de Biología Agrícola de Mendoza (IBAM-CONICET), Facultad de Ciencias Agrarias, Universidad Nacional de Cuyo, Mendoza, Argentina
b
Instituto de Química de San Luis (INQUISAL-CONICET), Facultad de Química, Bioquímica y Farmacia, Universidad Nacional de SanLuis, San Luis, Argentina
c
Instituto Nacional de Tecnología Agropecuaria (INTA-Bariloche), Estaci
on Experimental Agropecuaria Bariloche, CONICET, Río Negro, Argentina
article info
Article history:
Received 9 April 2015
Received in revised form
4 June 2015
Accepted 8 June 2015
Available online 12 June 2015
Keywords:
Arabidopsis thaliana
Germination
Melatonin
Senescence
UHPLC-MS/MS
abstract
Since the discovery of melatonin in plants, several roles have been described for different species, organs,
and developmental stages. Arabidopsis thaliana, being a model plant species, is adequate to contribute to
the elucidation of the role of melatonin in plants. In this work, melatonin was monitored daily by UHPLC-
MS/MS in leaves, in order to study its diurnal accumulation as well as the effects of natural and artificial
light treatments on its concentration. Furthermore, the effects of exogenous application of melatonin to
assess its role in seed viability after heat stress and as a regulator of growth and development of
vegetative tissues were evaluated. Our results indicate that melatonin contents in Arabidopsis were
higher in plants growing under natural radiation when compared to those growing under artificial
conditions, and its levels were not diurnally-regulated. Exogenous melatonin applications prolonged
seed viability after heat stress conditions. In addition, melatonin applications retarded leaf senescence.
Its effects as growth promoter were dose and tissueedependent; stimulating root growth at low con-
centrations and decreasing leaf area at high doses.
©2015 Elsevier Masson SAS. All rights reserved.
1. Introduction
Melatonin (MT, N-acetyl-5-methoxytryptamine) was primarily
known as a vertebrate pineal secretory molecule (Reiter, 1991) and
was first isolated from bovine pineal gland by Lerner et al. (Lerner
et al., 1958). In 1991, melatonin was detected in Lingulodinium
polyedrum and, later, in other dinoflagellates and green algae
(Hardeland and Fuhrberg, 1996; Balzer and Hardeland, 1991). In
1995, Dubbels et al. and Hattori and colleagues submitted reports
showing the presence of melatonin in plants (Dubbels et al., 1995;
Hattori et al., 1995).
To date, the presence of melatonin has been demonstrated in
more than 20 dicotyledonous and monocotyledonous plant
families (Posmyk and Janas, 2009). Nearly 60 commonly used
Chinese medicinal herbs contain melatonin with a wide range of
concentrations from nanograms to micrograms per gram of plant
tissue (Chen et al., 2003). There is vast evidence that the presence of
melatonin in plants helps to protect them from oxidative damage
and from adverse environmental conditions (Hardeland and
Fuhrberg, 1996; Kolar and Machackova, 2005; Wang et al., 2013;
Lee et al., 2014). Alpine and Mediterranean plants exposed to UV
radiation showed an increase in melatonin contents, suggesting
that melatonin antagonizes the damage caused by light-induced
stress, probably through its antioxidant properties (Pandi-
Perumal et al., 2006).
Melatonin is rhythmically secreted by the pineal gland in ver-
tebrates and is involved in regulation of circadian and, sometimes,
seasonal rhythms (Reiter, 1993). Circadian rhythms with nocturnal
maxima have also been described for insects and for L. polyedrum
(Hardeland and Poeggeler, 2003; Poeggeler et al., 1991). Contrary to
these findings, the studies of daily variations of melatonin contents
in plants have displayed contrasting results. While experiments did
not show any clear pattern of changes in a 12 h light/12 h dark
regime in Pharbitis nil and Solanum lycopersicum (Van Tassel et al.,
Abbreviations: MT, melatonin; MS/MS, tandem mass spectrometry; UHPLC, ultra
high performance liquid chromatography; Fv/Fm, maximum quantum efficiency of
photosystem II photochemistry; PSII, photosystem II.
*Corresponding author. Instituto de Biología Agrícola de Mendoza (IBAM-
CONICET), Facultad de Ciencias Agrarias, Universidad Nacional de Cuyo, Almte.
Brown 500, CP5505, Chacras de Coria, Mendoza, Argentina.
E-mail address: msilva@fca.uncu.edu.ar (M.F. Silva).
Contents lists available at ScienceDirect
Plant Physiology and Biochemistry
journal homepage: www.elsevier.com/locate/plaphy
http://dx.doi.org/10.1016/j.plaphy.2015.06.011
0981-9428/©2015 Elsevier Masson SAS. All rights reserved.
Plant Physiology and Biochemistry 94 (2015) 191e196

2001), a daily rhythm with a sharp maximum at night and very low
levels during the day was found in shoots of Chenopodium rubrum
(Wolf et al., 2001). Melatonin levels also fluctuate during the day in
grapes and Eichornia crassipes, showing a higher concentration
level at pre-dawn in grapes and a peak during the late light phase in
Eichornia (Boccalandro et al., 2011; Tan et al., 2007).
Different functional roles for melatonin have been described in
both organs and developmental stages of several plant species. For
example, it has been described that melatonin protects Brassica
oleracea rubrum seeds and young seedlings against toxic concen-
trations of copper (Posmyk et al., 2008). Melatonin also plays a
function in cold resistance, improving cucumber germination
during chilling stress (Posmyk et al., 2009) and protecting cold-
stressed wheat (Turk et al., 2014) and rice seedlings (Kang et al.,
2010).
Previous studies have suggested that melatonin could act as a
growth promoting compound, probably increasing auxin levels or
showing an auxin-like activity (Chen et al., 2009). When etiolated
hypocotyls from Lupinus albus were treated with a range of mela-
tonin and indole-3-acetic acid (IAA) concentrations, both com-
pounds elicited plant growth at micromolar concentrations, but
repressed the growth at higher levels (Hern
andez-Ruiz et al., 2004).
It was also confirmed that melatonin acts as a growth promoter in
coleoptiles of wheat, barley, canary grass, and oat (Hern
andez-Ruiz
et al., 2005). Melatonin also affected the regeneration of lateral and
adventitious roots and the expansion of cotyledons in etiolated
seedlings of L. albus, and in Brassica juncea young seedlings. Lower
concentrations of melatonin have been found to stimulate the root
growth and to raise the endogenous levels of IAA, but higher con-
centrations have inhibitory effects (Chen et al., 2009; Hern
andez-
Ruiz et al., 2004).
Twenty years after the initial finding of melatonin in higher
plants, accurate determinations still represent a major challenge
because of plant (matrix)-specific problems (Kolar and
Machackova, 2005; Pape and Luning, 2006). Chemical complexity
of plant's extract can interfere with melatonin determinations,
giving false positive results if methods from vertebrate's melatonin
research are directly adopted, for example, because of coelution in
LC or cross-reactivity with antibodies of immunological methods
like RIA or ELISA (Pape and Luning, 2006). Setting-up more reliable
analytical methods for melatonin extraction, detection, and quan-
tification is a basic requirement to get more insight into the
melatonin roles in plant physiology and ecology (Caniato et al.,
2003).
In view of the molecular-genetic studies in plants, Arabidopsis is
the best resource to dissect the functions of genes responsible for
melatonin synthesis (Byeon et al., 2014; Lee et al., 2015). In this
work, optimal conditions for extracting melatonin from Arabidopsis
thaliana leaves prior to its determination by UHPLC-MS/MS were
established. Furthermore, melatonin levels were monitored during
the day as well at different growth conditions. In addition, the ef-
fects of exogenous application of melatonin to assess its role on
seed viability and as a regulator of growth and development of
plants were studied.
2. Materials and methods
2.1. Reagents and solutions
Melatonin was purchased from Sigma Chemical (St Louis, MO,
USA). Acetonitrile, dichloromethane, chloroform, ethyl acetate,
methanol, and water Optima
®
LC-MS grade were purchased from
Fisher Scientific (Fair Lawn, New Jersey, USA). Formic acid, 98%, was
obtained from Fisher Scientific (Loughborough, UK). HakaphosTM
was obtained from Compo (Spain). Ultrapure water (18 M
U
cm) was
obtained from EASY pure (RF Barnstead, IA, USA).
2.2. Plant materials and growth conditions
A. thaliana plants used for melatonin determination during the
day were cultivated at a greenhouse under natural radiation with a
12/12 h dark/light cycle, with a maximum light intensity of
800
m
mol/m
2
/s, at a constant temperature of 23 ±2

C. Leaves were
harvested every three hours and immediately frozen in liquid ni-
trogen and then lyophilized in darkness. Before the extraction,
lyophilized material was homogenized with liquid nitrogen with a
mortar and a pestle.
The concentrations of exogenous applied melatonin were
selected based on preliminary studies performed in our lab as well
as previous studies (Pelagio-Flores et al., 2012; Wang et al., 2012).
Plants used for the determination of leaf area and chlorophyll
fluorescence were grown in a growth-chamber with a photoperiod
of 12-h light/12-h darkness, light intensity of 180
m
mol/m
2
/s, and
temperature of 23

C. Plants were treated with 1 mL of the different
melatonin concentration solutions (10, 50, and 500
m
M) and with
distilled water (control) three times a week.
Plants used for root elongation determinations were germinated
and grown on Petri dishes containing MS medium (Murashige and
Skoog Basal Salts Mixture, catalog no. M5524, purchased from
Sigma Chemical (St. Louis, MO, USA)). Plants were placed in a
growth-chamber with a photoperiod of 12-h light/12-h darkness,
light intensity of 180
m
mol/m
2
/s, and temperature of 23

C.
2.3. Germination tests
Seeds were sterilized by chlorine fumes for 8 h, and air-dried
prior to melatonin treatment. Sterilized seeds were placed on 9-
cm diameter Petri dishes with 0.7% agar (Britania
®
) with and
without the addition of melatonin. The following melatonin con-
centrations were evaluated; 0 (control), 100, 300, 500, and
1000
m
M. Seeds were stratified for three days. After stratification,
seeds were placed in an oven at a constant temperature of 35

C for
three days and then germinated at 23

C in a growth chamber with
a 12-h light/12-h dark photoperiod. Ten replications of 25 seeds
were arranged in a completely randomized design. Seeds were
considered germinated when the seed coat was broken and the
radicle was visible.
2.4. Viability tests
For tetrazolium tests, seed coats were removed and then the
embryos were submerged in a 0.5% tetrazolium solution for 24 h at
35

C protected from light (Kristof et al., 2008). Red-stained em-
bryos were scored as viable embryos under an optical microscope
(Nikon Eclipse E200).
2.5. Leaf area and root length determinations
For measurements of leaf area, images of entire plants were
taken every seven days, from week 2e5, and the area was calcu-
lated using the program Measure (Datinf
®
). Root length was
determined using images taken at the end of the experiment (14
days).
2.6. Chlorophyll fluorescence
The chlorophyll fluorescence was measured using a MINIPAM
portable chlorophyll fluorometer (Heinz Walz GmbH, Effeltrich,
Germany). The maximum quantum efficiency of PSII
I.G. Hern
andez et al. / Plant Physiology and Biochemistry 94 (2015) 191e196192

photochemistry (Fv/Fm), the maximal fluorescence in the light-
adapted state (Fm), the nonphotochemical quenching of chloro-
phyll fluorescence (NPQ), the non-photochemical coefficient (qN),
and the photochemical quenching coefficient (qP) were the vari-
ables analyzed. Twenty minutes of dark adaptation with a leaf clip
was used to allow various photosynthetic and photoprotective
mechanisms and state transitions to relax.
2.7. Melatonin extraction
Melatonin extraction from leaves was carried out under dim
green light (2
m
mol/m
2
/s) to prevent analyte degradation. Ho-
mogenized tissues were accurately weighted (0.2 g), and trans-
ferred to a 15 mL glass tubes. After that, 1 mL of 50% (v/v)
methanolewater was added to each sample and then, tubes were
vortexed during 15 s. Ultrasonication was employed to assist and
accelerate the extraction of melatonin from vegetal tissues in an
ultrasonic bath (200 W, 15

C; Cleanson 1106, Buenos Aires,
Argentina) filled with cold water for 20 min. The supernatant was
decanted and centrifuged for 5 min at 3500 rpm (1852.2 g). The
glass tubes were stored at 4

C for 20 min and then 1 mL of 50% (v/
v) methanol ewater was added to each sample. The tubes were
vortexed until the pellet was resuspended and then, centrifuged
again for 5 min at 3500 rpm. The resulting supernatant was filtered
through a 0.22
m
m syringe filter (Osmonics
®
) and stored in an
amber vial suitable for UHPLC-MS/MS analysis.
2.8. Chromatographic conditions
An AcquityTM Ultra High Performance LC system (UHPLC)
(Waters, Milford, USA) equipped with autosampler injection and
pump systems (Waters, Milford, USA) was used. The autosampler
vial tray was maintained at 15

C. The needle was washed with
proper mixtures of acetonitrile and water. The separation was
performed by injecting 10
m
L of sample onto ACQUITY UHPLC
®
,C8
columns (Waters, Milford, USA) with 2.1 mm internal
diameter 50 mm length, and 1.7
m
m particle size. A mobile phase
gradient program with solvent A (formic acid, 0.1% (v/v)) and B
(acetonitrile, 0.1% (v/v) of formic acid) was applied at a flow rate of
0.25 mL min
1
. The gradient program started with 10% B, followed
by a linear increase of B to 50% in 3.0 min. Then the mobile phase B
was reduced to the initial conditions within 0.2 min, where it was
held for 0.8 min. Thus, the total chromatographic run time was
4.0 min. The column was kept at a 35

C temperature.
2.9. Mass spectrometry instrumentation and MS/MS conditions
Mass spectrometry analyses were performed in a Quattro
PremierTM XE Micromass MS Technologies triple quadrupole
mass spectrometer (MS/MS). ZSprayTM electrospray ionization
(ESI) source (Waters, Milford, USA) was operated in a positive
(ESþ)modeat350

C with N2 as the nebulizer gas and the source
temperature was kept at 150

C. The capillary voltage was main-
tained at 3.3 kV and the extractor voltage was set at 3.0 V. Ul-
trapure nitrogen was used as desolvation gas with a flow of
800 L h
1
. Argon was used as collision gas at a flow of
0.19 mL min
1
.
After optimization, detection was performed in MRM mode of
selected ions at the first (Q1) and third quadrupole (Q3). To
choose the fragmentation patterns of m/z(Q1) /m/z(Q3) for
the analyte in MRM mode, direct infusions (via syringe pump)
into the MS of melatonin standard solution in methanol was
performed and the product ion scan mass spectra was recorded.
Thus, the transitions: 233 >174 and 233 >216 were assessed.
Quantification of MT was done by measuring the area under the
specific peak using MassLinx Mass Spectrometry Software (Wa-
ters, Milford, USA).
2.10. Statistical analysis
Analysis of variance (ANOVA) and Bonferroni's post tests were
performed in order to assess minimum differences between means,
with a significance level of p 0.05. The analysis was carried out
with GraphPad Prism 5 software.
3. Results and discussion
3.1. Melatonin levels are not diurnally regulated in Arabidopsis
leaves
The UHPLC-MS/MS analyses were carried out following the
methodology previously developed in our lab (Gomez et al., 2013).
An optimization of the extraction strategy for melatonin in Arabi-
dopsis leaves was carried out for this work. Once the conditions for
extraction, separation, and quantification were optimized and
established, the methodology was applied to the determination of
melatonin by UHPLC-MS/MS in A. thaliana leaves that were har-
vested every three hours. Surprisingly, the melatonin content in
leaves did not show a significant variation during the day, and its
concentration was within 80e120 ng/g dry weight (Fig. 1). This
pattern was also observed and reported for morning glory (P. nil)
and tomato (S. lycopersicum) by Van Tassel et al. (Van Tassel et al.,
2001). On the contrary, there are some scientific studies reporting
daily variations in melatonin contents (Wolf et al., 2001;
Boccalandro et al., 2011; Tan et al., 2007; Kolar et al., 1997), but
showing dissimilar results. While Wolf et al. (Wolf et al., 2001)
couldn't associate the increased levels of MT with a direct action of
light in Chenopodium, Boccalandro et al. (Boccalandro et al., 2011)
observed a light-induced depletion of MT levels in Vitis, and Tan
et al. (Tan et al., 2007) reported high levels of MT near sunset in
Eichornia due to a promotion of MT synthesis by light. It is impor-
tant to mention that these results have been obtained not only in
different species, but also under several growth conditions.
3.2. Melatonin contents depends on growth conditions
The levels of melatonin in 5- week old plants growing both
under natural and artificial light conditions were quantified. It has
Fig. 1. Melatonin concentration (expressed as ng/g dry weight) at different times in
Arabidopsis thaliana leaves that were harvested every three hours. Data are shown as
the mean values of three replicates ±SE.
I.G. Hern
andez et al. / Plant Physiology and Biochemistry 94 (2015) 191e196 193

been reported that melatonin levels vary not only between
different species, but also within the various organs of the same
plant (Posmyk and Janas, 2009; Van Tassel et al., 2001). Melatonin
levels can also be affected by growth conditions (Tan et al., 2007;
Arnao and Hern
andez-Ruiz, 2013). In this study, it was found a
six-fold decrease of melatonin concentration in plants that were
cultivated in a growth-chamber with a 12-h light/12-h darkness
photoperiod, photosynthetically active radiation (PAR) of 180
m
mol/
m
2
/s, and temperature of 23

C when compared to plants cultivated
in a greenhouse under natural radiation with a maximum PAR of
800
m
mol/m
2
/s (at similar photoperiod and temperature to those
grown in chambers) (Fig. 2).
This significant difference in melatonin contents may be due to
the light intensity as well as the difference in spectral composition
between natural and artificial light treatments.
3.3. Melatonin maintains high levels of germination post heat stress
To investigate the effect of the application of melatonin on the
germination of seeds under stress, the germination percentage of
non treated and treated seeds with various melatonin concentra-
tions was evaluated. Melatonin treatment increased seed germi-
nation after the application of a heat stress up to a 60% when
compared to the control treatment (Fig. 3a). The highest level of
germination post stress was obtained with the 1000
m
MMT
treatment, reaching germination percentages of 92.8 ±2.1%. To
check whether the reduction in germination after heat stress was
due to the promotion of seed dormancy by warm temperatures or
to a decrease in seed viability, we analyzed seed viability of non-
germinating seeds by tetrazolium tests (Kristof et al., 2008). This
test revealed that most of the control seeds that had suffered heat
stress had lost their viability compared with melatonin treated
seeds (Fig. 3b). The latter demonstrates that melatonin can be used
to maintain high viability and germination of seeds that had suf-
fered heat stress, and this is probably due to its powerful antioxi-
dant capacity, which has been extensively verified in previous
reports (Poeggeler et al., 2002).
3.4. Melatonin maintains high levels of quantum efficiency of PSII in
post-flowering plants
The analysis of chlorophyll fluorescence has become one of the
most powerful and widely used methods for obtaining information
about the state of the photosystem (PSII). This technique reveals the
extent to which PSII uses the energy absorbed by chlorophyll and
how much it is damaged by an excess of light. The flow of electrons
through PSII is indicative of the overall rate of photosynthesis under
many conditions. The PSII is the part of the photosynthetic appa-
ratus most vulnerable to light-induced damage, which is often first
reflected in leaf status. In this study, maximum quantum efficiency
of PSII, estimated as the ratio of variable fluorescence (Fv) over the
maximum fluorescence value (Fm) for dark-adapted leaves, was
strongly reduced in non melatonin treated leaves after flowering.
The application of 500
m
M melatonin solution, enabled plants to
maintain higher Fv/Fm than the controls (Fig. 4). This data suggests
that MT can delay natural senescence of A. thaliana leaves in post-
flowering plants, probably due to its antioxidant effect. Similar
results had been reported in barley (Arnao and Hern
andez-Ruiz,
2009) and apple (Wang et al., 2012).
Fig. 2. Melatonin concentration (expressed as ng/g dry weight) in Arabidopsis thaliana
plants that were cultivated under different growth conditions. Data are shown as the
mean values of at least six replicates þSE. Asterisk (*) indicates significant differences
(P<0.0001).
Fig. 3. a Effect of exogenous melatonin on Arabidopsis thaliana seeds germination after
heat stress. Data are shown as the mean values of ten replicates þSE. Different letters
indicate significant differences between treatments according to Bonferroni's multiple
comparison tests (P<0.05). b Embryos of Arabidopsis thaliana seeds after tetrazolium
treatment observed under a microscope where A) non stressed seed (control); B)
stressed seed without the addition of melatonin and C) stressed seed embedded with
melatonin (1000
m
M). Experimental conditions as described in Section 2.3.
Fig. 4. Effect of melatonin on Fv/Fm in pre and post-flowering Arabidopsis thaliana
leaves. Data are shown as the mean values of ten replicates þSE. Asterisk (*) indicates
significant differences between treatments and control according to Bonferroni's
multiple comparison test (P<0.05).
I.G. Hern
andez et al. / Plant Physiology and Biochemistry 94 (2015) 191e196194

3.5. Melatonin regulates root length
A possible role of melatonin as a root growth stimulator was also
studied. A significant difference in the effect of different concen-
trations of melatonin on the root growth of 14-d seedlings was
found. Treatment carried out with 0.1
m
M melatonin increased root
elongation, while melatonin concentrations of 500
m
M and
1000
m
M had a significant inhibitory effect (Fig. 5). Similar results
have been reported in B. juncea (Chen et al., 2009), where it has also
been observed a root length promotion at 0.1
m
M melatonin con-
centration and a significant inhibitory effect in 2-d-old etiolated
seedlings at concentrations of 100
m
M. Pelagio-Flores also reported
an increase of rooth growth in A. thaliana, although these effects
were observed at higher concentrations than the ones reported
here (150
m
M) and in different growth conditions: photoperiod of
16-h light/8-h darkness, light intensity of 100
m
mol/m
2
/s, and
temperature of 22

C, without noticing any inhibitory effect
(Pelagio-Flores et al., 2012). However, inhibitory effects at low
concentrations of melatonin has also been reported in some
monocots species (Hern
andez-Ruiz et al., 2005).
3.6. High concentrations of melatonin reduce leaf area per plant
Leaf area is an important physiological feature for certain edible
plants such as lettuce, spinach, tea, tobacco, etc. Leaf area differ-
ences can be attributed to photosynthetic, water or nutrient de-
mands. Our results indicate that the application of exogenous
melatonin (500
m
M) significantly reduced leaf area, when
compared to controls (Fig. 6). These data may suggest a decrease in
growth-related gene expression according to the data obtained
recently by Weeda et al. (Weeda et al., 2014).
4. Conclusions
Several conclusions arise from our findings. We demonstrated
that melatonin contents in Arabidopsis depend on growth condi-
tions, but they are not diurnally regulated. This information could
be used for food industry (i.e. to improve food quality and plant
traits of agronomic interest such as an increment of aerial biomass
of green edible plants, or to improve the conditions for seed stor-
age). Exogenous melatonin is a potential plant bio-stimulator,
prolonging seed viability after heat stress as well as delaying
post-flowering leaf senescence. Thus, its application in agriculture
could result in a good, feasible and cost-effective method, pre-
venting stress and improving plant growth and development under
different conditions.
This paper provides useful data for further related research on
melatonin role in plants. However, taking into account that mela-
tonin effects as growth promoter are dose and tissueedependent;
more studies including different species at several developmental
stages are needed to establish its optimal concentrations to
enhance plant production.
Contributions
IGH, FJVG, SC, MVA and MFS designed the research. IGH and
FJVG performed most of the experiments. IGH, SC and MFS con-
ducted the data analysis. IGH, FJVG and MFS wrote the manuscript.
All the authors contributed to improving the paper and approved
the final manuscript.
Acknowledgments
The authors would like to gratefully acknowledge the financial
support received from CONICET (PIP 309), University of Cuyo (6/
A445), and National University of San Luis (INQUISAL) (PROICO
21512).
References
Arnao, M.B., Hern
andez-Ruiz, J., 2009. Protective effect of melatonin against chlo-
rophyll degradation during the senescence of barley leaves. J. Pineal Res. 46,
58e63.
Arnao, M.B., Hern
andez-Ruiz, J., 2013. Growth conditions influence the melatonin
content of tomato plants. Food Chem. 138, 1212e1214.
Balzer, I., Hardeland, R., 1991. Photoperiodism and effects of Indoleamines in a
Unicellular Alga, Gonyaulax polyedra. Science 253, 795e797.
Boccalandro, H.E., Gonz
alez, C.V., Wunderlin, D.A., Silva, M.F., 2011. Melatonin levels,
determined by LC-ESI-MS/MS, fluctuate during the day/night cycle in Vitis
vinifera cv Malbec: evidence of its antioxidant role in fruits. J. Pineal Res. 51,
226e232.
Byeon, Y., Lee, H.Y., Lee, K., Back, K., 2014. Caffeic acid O-methyltransferase is
involved in the synthesis of melatonin by methylating N-acetylserotonin in
Arabidopsis. J. Pineal Res. 57, 219e227.
Caniato, R., Filippini, R., Piovan, A., Puricelli, L., Borsarini, A., Cappelletti, E.M., 2003.
Melatonin in plants. Adv. Exp. Med. Biol. 527, 593e597.
Chen, G., Huo, Y., Tan, D.-X., Liang, Z., Zhang, W., Zhang, Y., 2003. Melatonin in
Chinese medicinal herbs. Life Sci. 73, 19e26.
Fig. 5. Effect of melatonin on root length of 14 days old Arabidopsis thaliana seedlings.
Data are shown as the mean values of at least thirty replicates þSE. Asterisks (*)
indicate significant differences between treatments and control according to Bonfer-
roni's multiple comparison test (P<0.05).
Fig. 6. Effect of melatonin on leaf area of Arabidopsis thaliana enriched with 1 mL of
melatonin solutions three times a week. Data are shown as the mean values of at least
ten replicates ±SE. Asterisks (*) indicate significant differences between treatments
and control according to Bonferroni's multiple comparison test (P<0.05).
I.G. Hern
andez et al. / Plant Physiology and Biochemistry 94 (2015) 191e196 195

Chen, Q., Qi, W.-b., Reiter, R.J., Wei, W., Wang, B.-m., 2009. Exogenously applied
melatonin stimulates root growth and raises endogenous indoleacetic acid in
roots of etiolated seedlings of Brassica juncea. J. Plant Physiol. 166, 324e328.
Dubbels, R., Reiter, R.J., Klenke, E., Goebel, A., Schnakenberg, E., Ehlers, C.,
Schiwara, H.W., Schloot, W., 1995. Melatonin in edible plants identified by
radioimmunoassay and by high performance liquid chromatography-mass
spectrometry. J. Pineal Res. 18, 28e31.
Gomez, F.J.V., Hern
andez, I.G., Martinez, L.D., Silva, M.F., Cerutti, S., 2013. Analytical
tools for elucidating the biological role of melatonin in plants by LC-MS/MS.
Electrophoresis 34, 1749e1756.
Hardeland, R., Fuhrberg, B., 1996. Ubiquitous melatonin: presence and effects in
unicells, plants and animals. Trends Comp. Biochem. Physiol. 2, 25e45.
Hardeland, R., Poeggeler, B., 2003. Non-vertebrate melatonin. J. Pineal Res. 34,
233e241.
Hattori, A., Migitaka, H., Iigo, M., Itoh, M., Yamamoto, K., Ohtani-Kaneko, R.,
Hara, M., Suzuki, T., Reiter, R.J., 1995. Identification of melatonin in plants and its
effects on plasma melatonin levels and binding to melatonin receptors in ver-
tebrates. Biochem. Mol. Biol. Int. 35, 627e634.
Hern
andez-Ruiz, J., Cano, A., Arnao, M.B., 2004. Melatonin: a growth-stimulating
compound present in lupin tissues. Planta 220, 140e144 .
Hern
andez-Ruiz, J., Cano, A., Arnao, M.B., 2005. Melatonin acts as a growth-
stimulating compound in some monocot species. J. Pineal Res. 39, 137e142.
Kang, K., Lee, K., Park, S., Kim, Y.S., Back, K., 2010. Enhanced production of melatonin
by ectopic overexpression of human serotonin N-acetyltransferase plays a role
in cold resistance in transgenic rice seedlings. J. Pineal Res. 49, 176e182.
Kolar, J., Machackova, I., 2005. Melatonin in higher plants: occurrence and possible
functions. J. Pineal Res. 39, 333e341.
Kolar, J., Machackova, I., Eder, J., Prinsen, E., van Dongen, W., van Onckelen, H.,
Illnerova, H., 1997. Melatonin: occurrence and daily rhythm in Chenopodium
rubrum. Phytochemistry 4 4, 1407e1413.
Kristof, J.R., Coppersmith, J.L., Hong, K., Liu, P.-P., Homrichhausen, T.M., Sun, J.,
Martin, R.C., Nonogaki, H., 2008. An Arabidopsis thaliana embryo arrest mutant
exhibiting germination potential. Seed Sci. Res. 18, 55e65.
Lee, H.Y., Byeon, Y., Back, K., 2014. Melatonin as a signal molecule triggering defense
responses against pathogen attack in Arabidopsis and tobacco. J. Pineal Res. 57,
262e268.
Lee, H.Y., Byeon, Y., Tan, D.X., Reiter, R.J., Back, K., 2015. Arabidopsis serotonin N-
acetyltransferase knockout mutant plants exhibit decreased melatonin and
salicylic acid levels resulting in susceptibility to an avirulent pathogen. J. Pineal
Res. 58 (3), 291e299.
Lerner, A.B., Case, J.D., Takahashi, Y., Lee, T.H., Mori, W., 1958. Isolation of melatonin,
the pineal gland factor that lightens melanocytes. J. Am. Chem. Soc. 80, 2587.
Pandi-Perumal, S.R., Srinivasan, V., Maestroni, G.J.M., Cardinali, D.P., Poeggeler, B.,
Hardeland, R., 2006. Melatonin. FEBS J. 273, 2813e2838.
Pape, C., Luning, K., 2006. Quantification of melatonin in phototrophic organisms.
J. Pineal Res. 41, 157e165.
Pelagio-Flores, R., Mu~
noz-Parra, E., Ortiz-Castro, R., L
opez-Bucio, J., 2012. Melatonin
regulates Arabidopsis root system architecture likely acting independently of
auxin signaling. J. Pineal Res. 53, 279e288.
Poeggeler, B., Balzer, I., Hardeland, R., Lerchl, A., 1991. Pineal hormone melatonin
oscillates also in the dinoflagellate, Gonyaulax polyedra. Naturwissenschaften
78, 268e269.
Poeggeler, B., Thuermann, S., Dose, A., Schoenke, M., Burkhardt, S., Hardeland, R.,
2002. Melatonin's unique radical scavenging properties eroles of its functional
substituents as revealed by a comparison with its structural analogs. J. Pineal
Res. 33, 20e30.
Posmyk, M.M., Janas, K.M., 2009. Melatonin in plants. Acta Physiol. Plant. 31, 1e11.
Posmyk, M.M., Kuran, H., Marciniak, K., Janas, K.M., 2008. Presowing seed treatment
with melatonin protects red cabbage seedlings against toxic copper ion con-
centrations. J. Pineal Res. 45, 24e31.
Posmyk, M.M., Bałabusta, M., Wieczorek, M., Sliwinska, E., Janas, K.M., 2009.
Melatonin applied to cucumber (Cucumis sativus L.) seeds improves germina-
tion during chilling stress. J. Pineal Res. 46, 214e223.
Reiter, R.J., 1991. Pineal melatonin: cell biology of its synthesis and of its physio-
logical interactions. Endocr. Rev. 12, 151e180.
Reiter, R.J., 1993. The melatonin rhythm: both a clock and a calendar. Cell. Mol. Life
Sci. 49, 654e664.
Tan, D.X., Manchester, L.C., Di Mascio, P., Martinez, G.R., Prado, F.M., Reiter, R.J., 2007.
Novel rhythms of N1-acetyl-N2-formyl-5- methoxykynuramine and its pre-
cursor melatonin in water hyacinth: importance for phytoremediation. FASEB J.
21, 1724e1729 .
Turk, H., Erdal, S., Genisel, M., Atici, O., Demir, Y., Yanmis, D., 2014. The regulatory
effect of melatonin on physiological, biochemical and molecular parameters in
cold-stressed wheat seedlings. Plant Growth Regul. 1e14.
Van Tassel, D., Roberts, N., Lewy, A., O'Neill, S., 2001. Melatonin in plant organs.
J. Pineal Res. 31, 8e15.
Wang, P., Yin, L., Liang, D., Li, C., Ma, F., Yue, Z., 2012. Delayed senescence of apple
leaves by exogenous melatonin treatment: toward regulating the ascorbatee-
glutathione cycle. J. Pineal Res. 53, 11e20.
Wang, P., Sun, X., Li, C., Wei, Z., Liang, D., Ma, F., 2013. Long-term exogenous
application of melatonin delays drought-induced leaf senescence in apple.
J. Pineal Res. 54, 292e302.
Weeda, S., Zhang, N., Zhao, X., Ndip, G., Guo, Y., Buck, G.A., Fu, C., Ren, S., 2014.
Arabidopsis transcriptome analysis reveals key roles of melatonin in plant de-
fense systems. PloS One 9, e93462.
Wolf, K., Kolar, J., Witters, E., van Dongen, W., van Onckelen, H., Machackova, I.,
2001. Daily profile of melatonin levels in Chenopodium rubrum L. depends on
photoperiod. J. Plant Physiol. 158, 1491e1493.
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andez et al. / Plant Physiology and Biochemistry 94 (2015) 191e196196