Cent. Eur. J. Biol. • 9(11) • 2014 • 1117-1126
DOI: 10.2478/s11535-014-0330-1
Central European Journal of Biology
Involvement of melatonin applied to Vigna radiata
L. seeds in plant response to chilling stress
Research Article
Katarzyna Szafrańska1,*, Rafał Szewczyk2, Krystyna Maria Janas1
Department of Ecophysiology and Plant Development,
University of Lodz, Faculty of Biology and Plant Environmental Protection,
90-237 Lodz, Poland
1
Department of Industrial Microbiology and Biotechnology
University of Lodz, Faculty of Biology and Plant Environmental Protection,
90-237 Lodz, Poland
2
Received 25 February 2014; Accepted 07 April 2014
Abstract: The impact of melatonin (50 µM L-1) applied to Vigna radiata seeds by hydro-priming on phenolic content, L-phenylalanine ammonialyase (PAL) activity, MEL level, antioxidant properties of ethanol extracts as well as electrolyte leakage from chilled and re-warmed Vigna
radiata roots of seedlings were examined. Seedlings obtained from non-primed seeds, hydro-primed and hydro-primed with MEL were
investigated after 2 days of chilling and 2 days of re-warming. At 25°C, the level of MEL in roots derived from seeds hydro-primed with
MEL was 7-fold higher than in roots derived from non-primed seeds. However, the content of MEL significantly decreased in all variants
investigated after re-warming, in contrast to PAL activity and phenolic levels, which reached the highest values. The antioxidant capacity
of ethanol extracts from chilled and re-warmed roots, determined by ABTS+• assay, was correlated with phenolic content while the
reducing ability of these extracts, determined by the FRAP method, correlated with PAL activity. However, both were the highest in rewarmed roots with applied MEL, which was accompanied by a significant decline in electrolyte leakage. Taken together, results may
indicate that MEL can play a positive role in plant acclimation to stressful conditions and activation of phenolic pathway by this molecule.
Keywords: Antioxidant properties • Chilling stress • L-phenylalanine ammonia-lyase • Melatonin • Phenolics • Vigna radiata
© Versita Sp. z o.o.
Abbreviations:
ABTS
- 2,2’-azinobis-(3-ethylbenzothiazoline-6sulfonic acid);
AFMK -N1-acetyl-N2-formyl-5-methoxykynuramine;
AMK
- N1-acetyl-5-methoxykynuramine;
C
- control non-primed seeds;
c3OHM - cyclic 3-hydroxymelatonin;
CAD
- collisionally activated dissociation;
CE
- collision energy;
CEP
- collision cell entrance potential (V);
CUR
- nitrogen curtain gas (psi);
CXP
- collision cell exit potential (V);
DP
- declustering potential (V);
EC
- electrical conductivity;
EL
- electrolyte leakage;
EP
- entrance potential (V);
FCR
FRAP
GAE
GS1
GS2
H
H-MEL
Ihe
IS
MEL
MRM
PAL
ROS
TBARS
TEAC
TEM
TPTZ
- Folin-Ciocalteu reagent;
- ferric reducing/antioxidant power;
- gallic acid equivalent;
- nebulizer gas (psi);
- heating gas (psi);
- hydro-primed seeds;
- seeds hydro-primed with MEL solution;
- interface heater;
- ions spray voltage (V);
- melatonin (N-acetyl-5-metoxytryptamine);
- multiple reaction monitoring;
- L-phenylalanine ammonia-lyase;
- reactive oxygen species;
- thiobarbituric acid reactive substances;
- trolox equivalent antioxidant capacity;
- temperature of heating gas;
- 2,4,6- tripyridy-s-triazine.
* E-mail: [email protected]
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Melatonin and phenolic compounds in Vigna radiata L. roots
1. Introduction
Low temperature is a major factor limiting the productivity
and geographical distribution of chilling-sensitive plant
species. Plants grown in chilling conditions may exhibit
a loss of vigour and reduced growth rate. This may result
from the decline in the enzymatic activity, membrane
rigidification, electrolyte leakage, destabilization
of protein complexes, RNA secondary structure
stabilization and others. It is known that at least part of
these phenomena is affected by reactive oxygen species
(ROS), resulting in oxidative stress [1]. However, plants
possess several strategies to cope with the harmful
effects caused by ROS. Antioxidant enzymes as well as
low molecular weight antioxidants, such as glutathione,
proline, melatonin (MEL) or phenolic compounds have
been found to play a vital role in improving the tolerance
to chilling by protecting cellular membranes against the
deleterious effects of ROS [2,3].
N-acetyl-5-methoxytryptamine
(MEL)
is a ubiquitously present molecule, exhibiting pleiotropic
biological activities in species from bacteria to
mammals. In plants, MEL is considered to be involved
in the regulation of circadian rhythm [4] as well as in
many physiological processes, e.g. root and shoot
development [5], flowering, flower and fruit development
or delaying leaf senescence [6,7].
Many researchers have described the protective
role of MEL against environmental stresses, such as
heavy metals [4,8], cold [9,10], UV-B radiation [11] or
salt stress [12]. Antioxidant activity of MEL seems to
participate in: (i) the direct scavenging of free radicals,
(ii) stimulation of antioxidant enzymes, (iii) augmentation
of the activities of other antioxidants, (iv) protection of
antioxidant enzymes against oxidative damage, or (v) an
increase in the efficiency of the mitochondrial electron
transport chain, thereby lowering electron leakage and
the generation of free radicals [6]. The MEL molecule,
a natural electron donor, is particularly effective in ROS
detoxification for two reasons: (i) it has a high affinity and
selectivity for certain radicals, in particular the hydroxyl
radical (OH•), and (ii) the ability to generate a scavenger
cascade, in which the primary and secondary products
formed by radical reaction contribute to the elimination
of radicals [13].
A large group of phenolics widely described in the
literature [14], accumulating in stress-affected tissues,
can play a specific role in the defence against pathogens
and herbivores, UV screening, energy dissipation, radical
scavenging, and in cell wall reinforcement [15]. These
compounds act as effective antioxidants due to their
redox properties allowing them to operate as reducing
agents, hydrogen donors and singlet oxygen quenchers
[16]. Phenolics are derived from trans-cinnamic acid,
which is formed by deamination of L-phenylalanine,
catalyzed by L-phenylalanine ammonia-lyase (PAL,
EC 4.3.1.5). The activity of this enzyme is affected by
various biotic and abiotic stresses including chilling
[17,18].
Some pre-sowing techniques of seed treatment
allow plants to germinate and grow under adverse
environmental conditions. Seed priming allows to adjust
the water content in the seed, either by soaking the
seeds in water (hydro-priming) or in low water potential
solutions (osmo-priming) to start metabolic processes
but preventing the initiation of the embryonic root
growth. These techniques are one of the physiological
methods improving seed performance and providing
faster and synchronized germination. Seed priming can
be combined with the application of plant hormones [19]
and biostimulators (e.g., proline [20] and MEL [8,21]).
The objective of this study was to determine the effects
of H and H-MEL on MEL and phenolic levels and PAL
activity as well as on membrane integrity and antioxidant
activity of extracts from roots of chilled and re-warmed
seedlings.
2. Experimental Procedures
2.1 Plant material
Mung bean (Vigna radiata L.) seeds obtained from
Szydelscy Gardening Company (Szczecin, Poland)
were hydro-primed with water (H) and 50 µM L-1 MEL
water solution (H-MEL), while non-primed seeds were
used as a control (C). The seeds were surface sterilised
with a fungicide (Thiuram, Organica-Sarzyna, Poland)
and placed in plastic boxes with cotton wool wetted with
distilled water and germinated at 25°C for 3 days. Then
the seedlings were divided into 2 groups: one of them
was exposed to 5°C for 2 days (5 C), the other was
kept under optimal conditions (25°C) (25 C). Chilled
seedlings were placed back to 25°C for 2 days (5C+Reg)
for evaluation of biochemical changes in roots caused
by chilling [22].
2.2 Hydro-priming of seeds
Hydro-priming was performed according to Taylor et al.
[23], using distilled water and MEL water solution at a
concentration of 50 µM L-1. The initial seed water content
(MC, moisture content) was calculated as a percentage
of fresh weight basis [24]: MC = (FW-DW)/FW x 100%.
First, fresh seeds were weighed (FW, fresh weight)
and then after 2 days of drying at 100°C, the dry mass
of seeds (DW, dry weight) was weighed. To establish the
final seed water content, seeds were imbibed in different
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PEG 8000 solutions giving osmotic potential between -1
and -2 MPa at 25°C [25]. After 5 days of imbibition with
PEG -1.5 MPa Vigna radiata seeds did not germinate,
so their final moisture (MCf, calculated like MCi) was
chosen as sufficient for pre-germinative metabolic
activity to occur while preventing radical emergence.
Hydro-priming involves the application of a strictly
controlled quantity of water to the seeds which was
determined according to the formula: WH O = Wti (MCf
2
– MCi) / (100 - MCf) [23]. Calculated volume of water
and MEL solution was added portionwise over 6 hours
using ROTATOR DRIVE STR4 (BioCote company).
The seeds were then dried at room temperature for 3
days and used for the experiments.
2.3 Melatonin extraction and assay
Melatonin extraction was done at 4°C according to the
modified methods of Guerrero et al. [26] and HernandezRuiz et al. [27]. Lyophilized V. radiata roots (0.3 g) were
homogenized with chilled K-phosphate buffer (pH 8.0),
supplemented with EDTA and BHT, shaken horizontally
(darkness/room temperature/15 min) and centrifuged
(20 000 x g/5°C/10 min). The supernatant was filtered
through 2 layers of Miracloth and after the addition of
ethyl acetate followed by 15 min of shaking, the first
organic phase was collected. The remaining aqueous
phase was adjusted to pH 3.0 with 1N H3PO4, and
after addition of the next dose of ethyl acetate followed
by 30 min of shaking, the second organic phase
was collected. The combined organic phases were
evaporated to dryness in a vacuum evaporator (RV06ML, Ika, Germany) and the pellet was dissolved in 1 mL
of deionized water. The purified extract was filtered by
bacterial filters (ISO-Disc Filters PTEF-4-2, 4 mm x
0.2 µm) and then subjected to HPLC-MS/MS analysis.
2.4 HPLC-MS/MS analysis of melatonin
Melatonin was analyzed using an Agilent 1200 LC
System coupled with AB Sciex 3200 QTRAP mass
detector equipped with TurboSpray Ion Source (ESI).
Each sample was injected onto Agilent SB-C18 column
(1.8 µm, 4.6 x 50 mm). Mobile phase A consisted of
2mM L-1 ammonium formate containing 0.1% formic
acid; mobile phase B consisted of 2mM L-1 ammonium
formate in acetonitrile containing 0.1% formic acid.
Aliquots of the tested samples (10 µl) were injected on
a column maintained in isocratic conditions A:B – 20:80,
temperature 40°C and 600 µl min-1 flow. The retention
time of MEL was 1.6 min. MS/MS detection was done
in MRM positive ionization mode. Optimized MRM pairs
for MEL were: 233.2-174.2 m/z (CE=17) – quantifier
ion, 233.2-130.1 (CE=57) – qualifier ion. The other
parameters of the detector were: CUR: 35.00; TEM:
600.00; GS1: 50.00; GS2: 60.00; Ihe: ON; IS: 5500.00;
CAD: Medium; DP: 36.00; EP: 5.50; CEP: 14.00; CXP:
3.00. The criteria for limit of detection (LOD) and limit of
quantitation (LOQ) were established by signal-to-noise
(S/N) determination with the following criteria: LOD≥5
and LOQ≥10. LOD and LOQ for MEL reached 0.06 ng
mL-1 (S/N=6.1) and 0.1 ng mL-1 (S/N=11.2), respectively.
The working range for the quantitation covered the
linearity of the standard curves from 0.1 ng mL-1 to
100 ng mL-1 (r=0.9992).
2.5 Electrolyte leakage test
The electrolyte leakage (EL) was measured to evaluate
the degree of cold injury in V. radiata roots [28].
The roots (0.5 g) were washed 3 times in deionized
water, weighed and immediately transferred to glass
tubes containing 10 mL of deionized water (ECo).
After 1h of incubation at 25°C, electrical conductivity
was measured (EC1), then samples were boiled for 10
minutes, cooled to 25°C and final conductivity (EC2) was
measured again using a conductivity meter (Seven Easy,
Mettler Toledo, Poland). The results were expressed
as a percentage of the total electrolyte leakage
% EL = 100 x (EC1-ECo) / (EC2-ECo).
2.6 L-phenylalanine ammonia-lyase (PAL)
extraction and assay
PAL activity was determined as described previously
Janas et al. 2000 [17]. The final supernatant was used
as a source of crude enzyme and for protein content
determination [29]. One unit of the enzyme activity
(1 mU) is defined as the amount of enzyme required
for the formation of 1 µmole of trans-cinnamic acid per
1 minute.
2.7 Total phenolic content
Fresh roots (0.5 g) were extracted by 5 mL of 96%
Et-OH and the total phenolic contents were determined
using a modified Folin-Ciocalteu colorimetric method
[22]. Total phenolics were calculated from the gallic acid
standard curve and expressed as milligram gallic acid
equivalents per gram of fresh weight (mg GAE g−1 FW).
2.8 Antioxidant activity determinations
2.8.1 ABTS•+ decolorisation assay
ABTS•+ radicals were generated by 12 h of incubation
with oxidizing agent (K2O8S2) (room temp. /
darkness). The working solution of ABTS•+ diluted with
Na-phosphate buffer, pH 7.4 was prepared for each
assay. Its absorbance at 732 nm was measured (A0) and
30 s after root extract addition (prepared as in section
2.7) the absorbance was measured again (A30’’). Based
on the loss of ABTS•+ the antioxidant capacity of ethanol
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Melatonin and phenolic compounds in Vigna radiata L. roots
extracts expressed as Trolox equivalent antioxidant
capacity per gram of fresh weight (µM TEAC g-1 FW)
was estimated [30]. The Trolox solution ranging from
5 to 30 µM L-1 was used for the standard curve preparation.
2.8.2. Ferric Reducing/Antioxidant Power (FRAP) assay
Briefly, the FRAP reagent contained 2.5 mL 10 mM L-1
TPTZ solution in 40 mM L-1 HCl plus 2.5 mL 20 mM L-1
FeCl3 and 25 mL 0.3 M L-1 acetate buffer, pH 3.6 was
prepared freshly and warmed at 37°C. After addition of
root ethanol extracts (prepared as in section 2.7) and
incubation at 37°C for 5 min, absorbance of the reaction
mixture was measured at 593 nm. The final result was
expressed as the concentration of antioxidants having
a ferric reducing ability equivalent to that of 1 mM L-1
FeSO4 based on the standard curve for FeSO4 x 7H2O
at a concentration range between 100 and 1000 µM L-1
[31].
2.9 Statistical analyses
The results are the mean values of three independent
experiments in three replications ± SD.
For all analyses, the Student’s test distribution criteria
were used. Differences at P < 0.05 were considered
significant.
3. Results and Discussion
3.1 MEL contents
Since MEL was discovered in plants [32,33] its
presence in all organs has been repeatedly described.
MEL content varies significantly and ranges from picoto micrograms per gram of plant material, reaching
highest concentrations in the seeds and leaves, while
lowest in fruit [34]. This indolamine is absorbed by plant
roots, as in the water hyacinth (Eichornia crassipes L.),
and delivered to leaves, where its level dramatically
rises [35]. Not only are roots capable of absorbing MEL
in a dose-dependent manner, but also the cotyledons
of lupin (Lupinus alba L.), leaves of barley (Hordeum
vulgare L.) and osmo-primed seeds of cucumber
[10,36,37]. Thus, although it is known that endogenous
MEL occurs in plant tissues, nothing is known about its
accumulation in seedling organs after priming the seeds
with exogenous MEL. Therefore, firstly we determined
MEL content in V. radiata roots under optimal conditions,
and then again after chilling and re-warming.
The level of MEL was highest in the roots of
seedlings derived from non-primed seeds (C) at 25°C
(0.237 ng g-1 DW), and slightly decreased after chilling
(0.180 ng g-1 DW), reaching the lowest level after
re-warming (0.090 ng g-1 DW) (Figure 1). A lower level
of MEL was observed in H roots at 25°C (0.130 ng g-1
DW) compared to C roots. When H roots were chilled
and re-warmed, MEL concentration was similar as in
C roots. Even though priming of V. radiata seeds with
50 µM L-1 MEL solution resulted in almost 7-fold increase
in MEL content (1.391 ng g-1 DW), as compared to C
roots, the profiles of MEL content decline in H-MEL and
C roots were very similar after chilling (25% and 24%,
respectively) and slightly different after re-warming (62%
and 78%, respectively) in comparison with the roots of
seedlings grown continuously at 25°C. These results
may suggest that both endo- and exogenous MEL can
be similarly metabolized in these conditions. The lower
level of MEL in chilled and re-warmed roots may also be
associated with the transport of MEL to the upper parts
of the seedlings; however, in order to determine this,
more detailed studies are needed. To our knowledge,
this is the first report on the content of MEL in plant roots
after seed priming with MEL solutions, thus it is difficult
to compare our results with the studies of other authors.
3.2 Determination of cell membrane damage
Chilling stress decreases membrane fluidity due to
the unsaturation of membrane lipids fatty acids, and
changes the composition and proportion of lipids and
proteins in the cell membrane [38]. Structural changes
in membranes elevate their permeability to ions and
water, which eventually disrupt the normal course of
physiological processes in the cell [39]. In order to
estimate the deterioration of membranes in the roots
examined, the electrolyte leakage test was performed.
The lowest % of electrolyte leakage was noticed in C
roots grown at 25°C, whereas it increased after chilling
and re-warming. An increase in % of electrolyte leakage
Figure 1.
Melatonin concentration in roots of Vigna radiata seedlings
derived from C, H and H-MEL seeds, which were grown
for 2 days at 25°C (25C) or at 5°C (5C) and then rewarmed for 2 days at 25°C after chilling (5C+Reg).
A description of the symbols (*, **, ***) as in Table 1.
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by about 1.5-fold relative to C roots was found in H
roots (Table 1). Our results provide indirect evidence
that rapid seed water uptake during hydro-priming
induced oxidative stress, which has been previously
demonstrated by increasing TBARS content [22]. These
observations are consistent with those of Bailly et al.
[40] and may suggest that seed water uptake during
hydro-priming triggers large H2O2 production, causing
disorders in the functioning of the membrane. This effect
was maintained in both chilled and re-warmed H-roots,
suggesting the occurrence of irreversible changes in
membrane integrity, a phenomenon frequently observed
in chilling-sensitive tissues [41]. In H-MEL roots, % of
electrolyte leakage decreased after re-warming (Table 1),
which seems to confirm our previous suggestion about
a protective role of MEL when the stress factor is removed
[22]. In another study, shrinkage and disruption of plasma
membrane caused by chilling were almost completely
inhibited by MEL treatment in the suspension of carrot
cells, suggesting that MEL is also involved in the integrity
of membranes [9]. This indolamine might position itself
between the polar heads of polyunsaturated fatty acids
within the cell membranes, thus reducing lipid peroxidation
and maintaining their optimal fluidity [42,43]. A positive
dose-dependent impact of MEL on the membrane
integrity, lipid peroxidation products and electrolyte
leakage in water-stressed cucumber seedlings have
been previously demonstrated [28]. Our earlier study
showed the same level of TBARS in C roots at 25°C and
5°C, but it increased after re-warming. In roots treated
with H and H-MEL, chilling and re-warming triggered a
significant TBARS decrease but in H-MEL roots this
decline was even more evident [22]. This may suggest
that the application of MEL during seed hydro-priming
can help V. radiata plants to acclimate to chilling stress.
A similar effect of injury limitation by chilling in V. radiata
seedlings was observed by Posmyk and Janas [20] when
seeds were hydro-primed with proline. Both of these
compounds (MEL and proline) are known scavengers of
free radicals, thus the protection of cell membrane might
result from their antioxidative action. However, given the
significant decrease in MEL content in the roots after
re-warming of V. radiata seedlings, (Figure 1) it may be
assumed that other mechanisms can also be responsible
for the reduction of lipid peroxidation. We speculate that
MEL may be involved in phenolic metabolism, and thus it
can improve the antioxidant potential of plant cells under
stress conditions. This assumption can be based on the
results of Bajwa et al. [44] who have found that the upregulation of cold signalling genes by exogenous MEL may
stimulate the biosynthesis of cold-protecting compounds,
such as phenolics, and contribute to increased tolerance
of chilling stress. By preventing oxidative damage,
phenolics limit the production of TBARS, which is
beneficial for the functioning of cell membranes [22,45].
Aerobic organisms protect themselves against ROS by
synthesizing various secondary metabolites such as
MEL [28] or phenolics [17,18,22]. Therefore, in the next
experiments, we measured the level of total phenolics
and PAL activity.
3.3 PAL activity and total phenolic contents
PAL activity varies considerably between plant species
and shows developmental and tissue-specific variation.
Although stress factors usually increase the activity
of PAL, in the present work, chilling decreased this
parameter in all investigated variants (Figure 2), while
the content of phenolics was increased, particularly
in H-MEL roots (Figure 3). This was contrary to the
results obtained by Janas et al. [17], where the increase
in phenolic content in chilled soybean roots was
accompanied by increased PAL activity. With respect
to the lower activity of PAL and relatively higher levels
of soluble phenolics after chilling, a direct correlation
could not always be found [46]. It is possible that
under chilling stress, phenolics were not synthesized
de novo by pathways involving PAL activity but they
were constitutively present in the tissues. Thus these
compounds may be derived from pre-existing forms,
e.g., cellular structures or conjugates, which act as
a reservoir of aglycones and can be mobilized if needed
[18,47]. The mechanisms responsible for PAL activation
% of total electrolyte leakage
C
H
H-MEL
25°C
14.83 ± 3.37
*23.04 ± 0.32
18.23 ± 2.08
5°C
16.15 ± 1.55
*20.83 ± 0.80
**22.45 ± 1.37
5°C+Reg
18.10 ± 1.60
*21.70 ± 0.80
*15.30 ± 1.10
Table 1.
% of total electrolyte leakage (% EL) from roots of Vigna radiata seedlings derived from C, H and H-MEL seeds, which were grown for 2
days at 25°C (25C) or at 5°C (5C) and then re-warmed for 2 days at 25°C after chilling (5C+Reg). The symbols (*, **, ***) show that
results obtained for H and H-MEL are significantly different from C at 0.05, 0.01 and 0.001 level of Student’s test. Bars represent standard
deviations.
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Melatonin and phenolic compounds in Vigna radiata L. roots
in the cold are still poorly understood. PAL activity after
re-warming has increased in all variants investigated but
particularly in H-MEL roots (Figure 2), which can suggest
that PAL was not only activated by the damage caused
by chilling but also by the exogenous application of MEL.
Similar profiles of total phenolic content after re-warming
(Figure 3) may indicate that this significant phenolic
accumulation in roots of V. radiata was not induced by
chilling, but rather by the disappearance of the stressor,
similar to lettuce [48]. The results obtained in petunia
plants [49] and grapevine roots [50] recovered from
chilling as well as spectral analyses of ethanol extracts
from re-warmed roots of V. radiata [22] seem to confirm
this suggestion. Simultaneous increase in PAL activity
and phenolic content after re-warming (Figure 2, 3) can
imply that the accumulation of these compounds is PALdependent. It should be mentioned that the age of the
seedlings and their physiological stage may cause some
concerns about the experimental design, as at the end
of the 25°C and 5°C treatments, seedlings were 5-daysold (3 d at To + 2 d at 25 or 5°C), whereas 5C+Reg
seedlings were 2 days older (3 d at To + 2 d at 5 °C +
2 d at 25°C). Even if the increased activity of PAL and
the total phenolic content after re-warming (Figure 2, 3)
were related to aging rather than the response to chilling
stress, it is evident that H-MEL roots accumulated
much more of these compounds than C and H roots.
This may confirm our earlier hypothesis on the
participation of MEL in phenylpropanoid pathway
activation [22]. However, further studies are needed to
clarify this phenomenon, especially taking into account
the latest results of Shin et al. [51]. These authors
have found that myricetin, a natural bioflavonoid
extracted from medicinal plants, inhibits the activity of
AANAT (serotonin N-acetyltransferase (EC 2.3.1.8.7),
penultimate enzyme MEL biosynthesis) in the pineal
gland of rats, and thereby reduces the level of MEL
in the blood. Although the search for homologues of
the last two enzymes in MEL biosynthesis in plants is
still ongoing, it cannot be excluded that the increased
PAL activity (Figure 2) and elevated levels of phenolics
(Figure 3) in all re-warmed variants may not be the
result of stimulating action of MEL but rather a reason
for the decline in MEL level (Figure 1). On the other
hand, the structure and nature of these compounds can
have a considerable impact on their mode of action, and
therefore, perhaps both of these phenomena are not
mutually exclusive. Certainly, more research is needed
that would attempt to explain this observation.
Given the widely reported antioxidant properties of
phenolics and MEL in the literature, the next step of our
study was to investigate the antioxidant potential of root
ethanol extracts.
Figure 2.
Specific PAL activity in roots of Vigna radiata seedlings
derived from C, H and H-MEL seeds, which were grown
for 2 days at 25°C (25C) or at 5°C (5C) and then rewarmed for 2 days at 25°C after chilling (5C+Reg).
A description of the symbols (*, **, ***) as in Table 1.
Figure 3.
Total phenolic contents in roots of Vigna radiata seedlings
derived from C, H and H-MEL seeds, which were grown
for 2 days at 25°C (25C) or at 5°C (5C) and then rewarmed for 2 days at 25°C after chilling (5C+Reg).
The results are expressed as a GAE levels. A description
of the symbols (*, **, ***) as in Table 1.
3.4 Antioxidant properties of ethanol extracts
Antioxidant activity of phenolics is mainly caused by
their redox properties, which can play an important role
in adsorbing and neutralising free radicals, quenching
singlet and triplet oxygen or decomposing peroxides
[52]. Due to the complex nature of these compounds,
it is beneficial to evaluate this activity by at least two
complementary methods. In our study, ABTS+• and
FRAP assays were selected. Antioxidant properties
determined by ABTS+• assay showed a similar
tendency to that describing the content of phenolics.
Chilling stress increased antioxidant properties of all
3 variants, but after re-warming, the extracts from
H-MEL roots revealed the highest antioxidant potential
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(Figure 4). A close correlation between the increase in
antioxidant properties and higher phenolic content has
been already demonstrated in Petunia x hybrida [49].
These authors found that the re-warming increased
ROS scavenging ability, which was associated with a
higher amount of soluble phenolics similar to the present
study, particularly for H-MEL root extracts. One of the
most appealing properties of MEL, which distinguishes it
from most antioxidants, is the fact that MEL metabolites
also have the ability to scavenge ROS and reactive
nitrogen species [13]. The study of Tan et al. [53]
showed that a single MEL molecule can eliminate four
radicals in the interaction with ABTS•+ in a reaction chain
leading to the formation of active metabolites, such
as cyclic 3-hydroxymelatonin (c3OHM), N1-acetyl-N2formyl-5-methoxykynuramine (AFMK), and N1-acetyl5-methoxykynuramine (AMK), compounds that also
constitute efficient and versatile free radical scavengers
[54]. We cannot exclude that in our experiments
phenolics and metabolites of MEL are responsible for the
antioxidant properties of ethanol extracts from H-MEL
roots, especially as the level of MEL has significantly
declined after re-warming (Figure 1). It is likely that the
high antioxidant efficacy in this variant was the result
of the joint action of phenolics and MEL metabolites.
Vitalini et al. [55] considered the possibility that the
documented beneficial health properties of grape/wine
are not only due to the presence of phenolics, but also
MEL, its isomers and hundreds of other phytochemicals
present in these foods.
Measurement of the reducing potential could also be
an indicator of antioxidant activity, therefore, we have
chosen to use FRAP assay as the second method in
this work. We selected this method because it treats the
antioxidants in the samples as reductants in a redoxlinked colorimetric reaction. One possible disadvantage
of the FRAP assay is the fact that it does not evaluate the
antioxidant capacity of an agent, which does not have
the ability to reduce Fe3+ to Fe2+ such as glutathione.
The reducing power of MEL explains some aspects of
antioxidant activity and can be directly related to the
amount of phenolics [56]. In the current study, the ability
of compounds to reduce Fe3+ ions to Fe2+ (Figure 5)
differed from the scavenging properties measured by
the ABTS•+ method (Figure 4) and the results were not
consistent with the level of phenolics (Figure 3). It should
be noted that the assay measuring total phenolics is
conducted at rather basic conditions (pH 10, necessary
for phenols to dissociate protons), whereas FRAP assay
is carried out at acidic (pH 3.6) conditions. Although
simple phenols can react with FCR, they are not effective
radical scavenging antioxidants. Therefore, a significant
correlation between the total phenolic level and the
radical scavenging activity of a sample not always can
be found [57]. Randhir and Shetty [58] suggested in their
study that the antioxidant function of the mung bean
extracts depended on the qualitative characteristics of
phenolic profile and not just on the total phenolic content.
In our study, the reducing capacity of ethanol extracts
was rather correlated with PAL activity (Figure 2) and
showed a decline after chilling and a significant increase
after re-warming, especially in H-MEL roots (Figure 5).
This may suggest that the reducing power of extracts
was the result of PAL action and products synthesized
de novo in phenylpropanoid pathway. Our previous
results [22] proved that after re-warming of chilled
Figure 4.
Scavenging activity of ethanol extracts from roots of
Vigna radiata seedlings derived from C, H and H-MEL
seeds, which were grown for 2 days at 25°C (25C) or at
5°C (5C) and then re-warmed for 2 days at 25°C after
chilling (5C+Reg). The results are expressed as a TEAC
levels. A description of the symbols (*, **, ***) as in
Table 1.
Figure 5.
FRAP of ethanol extracts from roots of Vigna radiata
seedlings derived from C, H and H-MEL seeds, which
were grown for 2 days at 25°C (25C) or at 5°C (5C)
and then re-warmed for 2 days at 25°C after chilling
(5C+Reg). The results are expressed as FeSO4
equivalents. A description of the symbols (*, **, ***) as
in Table 1.
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Melatonin and phenolic compounds in Vigna radiata L. roots
V. radiata seedlings, the nature of phenolic deposits
accumulating in vacuoles were completely reversed in
comparison to those acquired after chilling. We cannot
exclude that the presence of MEL in H-MEL extracts
strongly contributed to their antioxidant properties via
phenylpropanoid pathway activation [44]. It is believed
that the action of most antioxidants is a combination of
mechanisms, and probably a similar phenomenon was
observed in the extracts studied in this work.
4. Conclusions
The results presented above provide correlative
evidence indicating that MEL applied to the seeds
of V. radiata can improve the defense mechanisms
of plants during cold acclimation by increasing the
activity of PAL after re-warming. This process, in
turn, is accompanied by the induced accumulation of
total phenolics and increased antioxidant capacity.
As the level of MEL rapidly decreased after re-warming,
enhanced chilling tolerance was rather caused by the
activation of phenylpropanoid pathway than direct
action of MEL. However, the mechanisms of tolerance
to chilling stress are complex and may act in concert
with other molecular, biochemical and physiological
mechanisms in order to maintain normal physiological
functions under chilling conditions. Further studies are
needed to examine the potential contribution of MEL
to phenylpropanoid pathway activation during cold
acclimation.
Acknowledgments
The research was supported by University of Lodz
(Poland) project No 505/398.
The authors would like to thank Magdalena Kuzaniak
and Magdalena Drązikowska for their help in carrying
out experiments.
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