Changes in melatonin content of pepper seeds during

storage
G. Yakupoğ lu1, a, Ş. Kö klü 2, A. Karaca2, E. Dü ver2, A. Klicic2 and A. Korkmaz2
1Bozok University, Yozgat, Turkey; 2Kahramanmaraş Sü tçü Iİmam University, Kahramanmaraş, Turkey.

Abstract
Melatonin (N-acetyl-5-methoxytryptamine) is present in evolutionary distant
organisms like bacteria, algae, invertebrates, vertebrates and plants. Melatonin may
serve as a photoperiodic and circadian rhythm regulator as well as a universal
antioxidant. Even though little is known about changes in seed melatonin content
during short-term storage, there is no information available on the involvement of
melatonin on seed aging and seasonal variation of seed melatonin content during long-
term storage. The main aim of this research was to identify the changes in melatonin
content of pepper seeds (‘Yalova 341’) stored for two years at two temperature regimes
and to determine the possible effects of melatonin on the seed aging process. For this
purpose, pepper seeds were treated with 0 or 250 µM melatonin for one day after which
they were stored for up to two years under room temperature (25°C) or in the
refrigerator (4°C). The effects of exogenous application of melatonin on seed aging and
changes in melatonin content were assessed periodically with various tests and
analyses. The results showed that seed melatonin content varied significantly during
storage, reaching its peak in October and bottoming out in August during the course of
the experiment. On the other hand, seed MDA and H2O2 contents were significantly
higher and lower in October and August, respectively, and melatonin pre-treatment
slightly improved seed germination performance during the course of the experiment.
These results clearly indicate that the existing circannual rhythm in melatonin levels
lasts longer than a year, presumably over the life of the seed albeit in a diminishing
fashion.

Keywords: germination, seed storage, seasonal change, seed aging

INTRODUCTION
Melatonin (N-acetyl-5-methoxytryptamine) is an indoleamine initially isolated for the
first time from the bovine pineal gland more than half a century ago (Lerner et al., 1958). Since
then, it was discovered in evolutionary distant organisms including bacteria, algae,
invertebrates, vertebrates and finally in higher plants (Arnao and Hernandez-Ruiz, 2015; Tan
et al., 2012).
Extensive research has been conducted on melatonin which was formerly known to be
a hormone existing exclusively in animals. Studies have shown that melatonin was indeed
present in every plant species tested and the main roles of melatonin are to regulate circadian
rhythms as well as growth and development and to enhance tolerance to various
environmental stress factors (Arnao and Hernandez-Ruiz, 2014). Evidence indicates that
plants living under stressful environments have a higher melatonin content compared to
those living under normal conditions and that exogenous application of melatonin can also
improve stress tolerance of plants that do not produce enough melatonin endogenously
(Arnao and Hernandez-Ruiz, 2015). Recent studies have documented that melatonin is a
proven powerful free radical scavenger and a wide spectrum antioxidant in plants (Tan et al.,
2012), and supplies important protection against such environmental stresses as low
temperature (Korkmaz et al., 2017), high temperature (Shi et al., 2015), salinity (Li et al.,
2012), water (Zhang et al., 2013) and excessive UV light (Afreen et al., 2006).

aE-mail: gokcenyakupoglu@gmail.com

Acta Hortic. 1273. ISHS 2020. DOI 10.17660/ActaHortic.2020.1273.55 425

XXX IHC – Proc. VIII Int. Symp. on Seed, Transplant and Stand Establishment
of Horticultural Crops
Eds.: D.I. Leskovar et al.
 Even though seed quality starts to decline while the seeds are still on the mother plant
and continues through harvest and storage until sowing in the field; most of the seed quality
loss occurs during storage (Bewley et al., 2013). Storing the seeds in an environment with
unfavorable conditions such as high relative humidity and temperature accelerates the aging
process due to the fact that reactive oxygen species formed in seeds oxidase the phospholipids
and fatty acids in membranes (Shaban, 2013). Most of the data on melatonin being a broad
spectrum antioxidant that enhances stress tolerance come from investigations conducted at
the whole plant level but very little data are available on seasonal fluctuations of seed
melatonin content and its involvement on seed viability or aging during long-term storage.
Therefore, this study was carried out to identify the possible effects of melatonin on pepper
seed aging process and the changes in melatonin content in pepper seeds stored for two years
at two temperature regimes.

MATERIALS AND METHODS

Plant material and melatonin treatments

Seeds of ‘Yalova 341’ pepper (Capsicum annuum L.) were purchased from Argeto Seed
Company, Turkey. Seed moisture content was determined according to ISTA (2010) rules and
found to be 6.15% on fresh weight basis. Single layers of pepper seeds (100 g) were placed in
20×30×5 cm (l×w×h) covered transparent plastic boxes between double layers of filter paper
moistened with 250 µM melatonin (Sigma-Aldrich, MO, USA) solution and kept at 25°C in
darkness for 24 h. The seeds were then rinsed for 1 min under running water and left to dry
back to original moisture content on paper towels under room conditions. After drying they
were sealed in individually laminated plastic bags covered with aluminum foil (5 g) and placed
at 4°C (refrigerator) or 25°C (incubator) for two years from the beginning of December 2015
to December of 2017. Untreated seeds having the same moisture content were also packaged
and stored under the same conditions as mentioned above.

Measurements and analyses

Seeds were sampled every two months starting from the beginning of storage to
determine possible seasonal changes in melatonin content and the effects of melatonin
application on seed aging during storage. At the end of each storage time, seed moisture
content was determined and following analyses were conducted.
To determine seed melatonin content 0.5 g seed was placed in test tubes containing
ethyl acetate (3 mL) following homogenization with liquid nitrogen. After leaving overnight
(17 h) at 4°C in darkness with shaking, the tubes with plant tissues were centrifuged at 6,000
g and 4°C for 20 min. The supernatant was transferred to another tube and the remaining
plant residue was washed with 0.5 mL ethyl acetate. The extract and washing from each
sample were evaporated to dryness under vacuum. The residue was re-dissolved in methanol
(0.5 mL), filtered (0.45 μm) and analyzed using HPLC with fluorescence detection. Extraction
of melatonin and HPLC analysis were always carried out under dim light by the method
reported in Korkmaz et al. (2014).
Germination tests were carried out in darkness in temperature-controlled incubators at
25±1°C. Seeds were disinfected in 1% (active ingredient) sodium hypochlorite solution for 10
min to eliminate possible seed-borne microorganisms, rinsed for 1 min under running water
prior to drying for 2 h at room temperature. Fifty seeds were placed on two layers of filter
paper moistened with 5 mL of distilled water in covered 9 cm Petri dishes. Treatments were
arranged in completely randomized design with four replications. Radicle protrusion to 2 mm
was scored as germination and germinated seeds were counted daily until the numbers
stabilized. From the total number of seeds germinated, final germination percentage (FGP)
and mean germination time (MGT) were calculated using Seed germination v. 1.0 software.
For the determination of malondialdehyde (MDA) content, 0.5 g seeds were
homogenized in 3 mL of 10% trichloroacetic acid (TCA) and centrifuged at 10,000 g for 15
min. The supernatant was collected and 1 mL was mixed with 1 mL of 0.6% thiobarbituric
acid. The mixture was boiled at 100°C for 20 min, cooled quickly, and centrifuged at 10,000 g

426
for 10 min after which its absorbance was measured at 532, 600, and 450 nm (Zhang et al.,
2005). The MDA concentration was calculated according to the following formula: MDA (µmol
g-1 FW) = 6.45×(A532–A600) – 0.56×A450.
To determine H2O2 content, seeds (0.5 g in 4 replicates) were homogenized in 3 mL of
1% (w/v) TCA. The homogenate was centrifuged at 10,000 g at 4°C for 10 min. Subsequently,
0.75 mL of the supernatant was mixed with 0.75 mL of 10 mM potassium phosphate buffer
(pH 7.0) and 1.5 mL of 1 M KI. H2O2 content of the supernatant was determined by comparing
its absorbance at 390 nm to a standard calibration curve and expressed as µmol g-1 FW (OÖ zden
et al., 2009).

RESULTS
Melatonin concentrations of untreated seeds and seeds treated with exogenous
melatonin showed similar significant seasonal changes regardless of storage temperature;
they reached their peaks and lowest levels around October and August, respectively (Figure
1). Additionally, in general, seeds stored at low temperature (4°C) had higher melatonin
concentrations than those stored at room temperature (25°C) at all storage times except in
the month of August. Physiological concentrations of melatonin in untreated seeds were quite
high (around 600 ng g-1 FW) at the beginning of the experiment but continuously declined,
reaching their lowest levels (2-3 ng g-1 FW) in August of 2016 regardless of storage
temperature. However, seed melatonin content increased significantly to170-225 ng g-1 FW in
October 2016 after which they declined again to their lowest values (1-2 ng g-1 FW) in August
of 2017. A similar trend in melatonin concentrations was also observed in the winter months
of 2017, which coincided with the end of the experiment. Seeds that were treated with
exogenous melatonin exhibited almost the same seasonal variation pattern during the course
of storage, peaking in October and reaching their lowest levels in August. Moreover, although
melatonin-treated seeds exhibited the same seasonal change in melatonin content as
untreated seeds, they always had considerably higher melatonin concentrations at all storage
periods (except August 2017) with the differences between treated and untreated seed
getting progressively smaller as the time in storage progressed.

Figure 1. Changes in melatonin levels of pepper seeds during 24-months of storage. Seeds
were treated with melatonin (0 and 250 µM), stored at 4°C and 25°C for two years
and sampled for melatonin analysis at 2-month intervals starting from December
of 2015.

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 The moisture content of the seeds rose considerably during the 2nd year of storage even
though seed were stored in sealed plastic packages after being dried back to their original
moisture content (Figure 2). Because the seeds were not stored hermetically, it is normal that
their moisture content increased over time in storage. Seed moisture changed slightly during
the first 6 months, rose sharply by the end of the first year and continued to increase steadily
until the end of the experiment, finally reaching around 7.4%. Seeds stored at 4°C had slightly
lower moisture content than those stored at 25°C at the end of the experiment.

Figure 2. Changes in moisture content of pepper seeds during 24-months of storage. Seeds
were treated with melatonin (0 and 250 µM), stored at 4°C and 25°C for two years
and sampled for moisture content analysis at 2-month intervals starting from
December of 2015.

Germination of seeds from all treatments changed slightly during the course of storage
and remained above 75% (Figure 3a). Storage temperature affected seed germination and
seeds stored at 4°C maintained slightly higher FGPs than those stored at 25°C until the end of
the experiment. Additionally, seeds treated with exogenous melatonin exhibited higher FGPs
during the two years in storage indicating that the melatonin treatment slowed the aging
process. Similarly, MGTs of all treatments increased steadily as the storage time progressed
and seeds stored at 25°C had higher MGT values than those stored at 4°C (Figure 3b)
especially toward the second year. Additionally, even though melatonin pre-treatment of seeds
did not cause any significant reductions in MGT in general, MGTs of all treatments were lower
in the month of October where seed melatonin contents rose significantly.
Seed H2O2 (Figure 4a) and MDA (Figure 4b) contents exhibited similar trends and
increased significantly during storage. However, it is interesting to note that strong peaks in
the concentrations of these two variables were observed in August of both years, coinciding
with the lowest levels of melatonin. Additionally, seeds treated with exogenous melatonin
generally had lower H2O2 concentrations than the untreated seeds and storing the seeds at
25°C resulted in significant rises in the amount of H2O2 present compared to storage at 4°C.
Seed MDA content exhibited a similar trend as well and seeds pre-treated with melatonin had
lower MDA contents in general compared to untreated seeds.

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Figure 3. Changes in final germination percentage (top) and mean germination time (MGT)
(bottom) of pepper seeds during 24-months of storage. Seeds were treated with
melatonin (0 and 250 µM), stored at 4 and 25°C for two years and subjected to
germination test at 2-month intervals starting from December of 2015.

DISCUSSION
To our knowledge, there is no information available related to long-term changes in
melatonin contents in pepper seeds. Herein, changes in the concentrations of these molecules
in pepper seeds stored for 2 years were established. Melatonin concentrations of untreated
seeds and melatonin-treated seeds showed similar significant seasonal fluctuations
regardless of storage temperature; they reached their peaks and lowest levels around October
and August, respectively. Additionally, seeds that were treated with exogenous melatonin
exhibited similar seasonal variation patterns during the course of storage, reaching peak
levels in October and lowest levels in August.

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Figure 4. Changes in H2O2 (top) and MDA (bottom) content of pepper seeds during 24-
months of storage. Seeds were treated with melatonin (0 and 250 µM), stored at 4
and 25°C for two years and sampled for MDA and H2O2 analysis at 2-month
intervals starting from December of 2015.

The possible role of melatonin in plants as a daily circadian rhythm regulator has been
well examined and the fluctuations in the melatonin concentration of plant tissues regarding
its possible role as a regulator of light/dark-mediated rhythms were associated with external
factors-photoperiod or temperature (Arnao and Hernandez-Ruiz, 2015). However, the results
of this study clearly show that seed melatonin concentrations exhibited significant seasonal
changes over the course of storage and that the changes are regulated by factors independent
of environmental conditions since seeds were stored in darkness and constant temperature
regimes. Similar seasonal change in seed melatonin content was observed only in corn and
cucumber seeds previously by a study conducted by Kołodziejczyk et al. (2015) where seed

430
melatonin content of these species varied significantly during 12-month storage, reaching
their peaks in winter months. However, to our knowledge, this is the first report that indicates
an existing circannual rhythm in melatonin levels in seeds; this rhythm possibly persists over
the life of the seed albeit in a diminishing fashion. Our results are in close agreement with
those of Kołodziejczyk et al. (2015) who suggested that there is a possible independent
‘chemical memory’ in plants which is not affected by extrinsic factors and that significant
seasonal variations in seed melatonin content may be indicative of an endogenous biological
clock operating as a biochemical calendar (possibly acquired through an evolutionary
process) precisely regulated by melatonin.
Melatonin pre-treatment of seeds slightly improved the germination performance of
seeds at both temperature conditions since seeds from all treatments exhibited 75% or higher
germination at the end of storage. This may be due to the fact that the seeds had really high
vigor at the beginning of experiment and retained this characteristic quite well until the end
of the experiment where it begun to decline. Even though there is no information about the
involvement of melatonin in seed aging, recent studies documented that melatonin
application enhanced germination performance of seeds under stressful conditions. For
example, Korkmaz et al. (2017) found that melatonin-treated pepper seeds exhibited
improved germination performance under chilling conditions. Additionally, pre-treatment
with melatonin boosted seed germination to 60% in Arabidopsis thaliana after exposure to
heat stress compared to the control treatment (Hernandez et al., 2015).
Peroxidation of lipids in cellular membranes caused by free radicals such as O2•-, •OH
and H2O2 results in impaired membrane function, reduced fluidity, and inactivation of
membrane-bound enzymes and receptors in aging seeds (Bewley et al., 2013). The
decomposition of polyunsaturated fatty acids in the membranes by peroxidation generates
MDA, a toxic byproduct; thus, determining free radical levels and MDA contents are valuable
tools. Seed H2O2 (Figure 4a) and MDA (Figure 4b) contents exhibited similar trends and
increased significantly during storage. However, it is interesting to see that strong peaks in the
concentrations of these two variables were observed in August of both years, coinciding with
the lowest levels of melatonin. The fact that higher seed MDA and H2O2 contents and lower
melatonin levels observed in August were followed by the lower MDA and H2O2 and the higher
melatonin levels in October support the findings of Zhang et al. (2013) who reported that
oxidative stress caused by high temperature caused the accumulation of MDA in cherry fruits,
triggering melatonin synthesis.

CONCLUSIONS
Long-term seasonal changes in melatonin contents in pepper seeds stored for 2 years
were established. The results indicate that seed melatonin content varied significantly over
the course of the two-year storage, reaching its peak in October and lowest levels in August.
Higher levels of MDA and H2O2 determined in seeds in August coincided with the lowest levels
of melatonin present in the seeds indicated that melatonin was directly involved in the
regulation of lipid peroxidation in seeds during the aging process. The results of this research
also indicate that pre-treating the seeds with melatonin before storage could be used to slow
the aging process in pepper seeds since it has a potential to improve the germination
performance of seeds stored under both temperature conditions.

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