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J. Pineal Res.

2005; 39:333341 Copyright  Blackwell Munksgaard, 2005


Doi:10.1111/j.1600-079X.2005.00276.x Journal of Pineal Research

MINI REVIEW
Melatonin in higher plants: occurrence and possible functions

Abstract: Melatonin may be ubiquitous in the plant kingdom. This review Jan Kolar and Ivana Machackova
considers the evaluation of methods of melatonin determination in plant Institute of Experimental Botany, Academy of
material and possible melatonin functions in plants. Concerning the Sciences of the Czech Republic, Prague,
determination methods, the only reliable techniques are liquid Czech Republic

chromatography mass spectrometry or gas chromatography mass


spectrometry after some purication steps of the extract. Melatonin was Key words: antioxidant, circadian rhythms,
shown to delay ower induction in some photoperiodic plants and in the higher plants, melatonin, melatonin
determination, photoperiodism, reactive
dinoagellate Lingulodinium it replaces, in part, the requirement of darkness
oxygen species
for cyst formation. Melatonin may also have a function as an antioxidant
Address reprint requests to Ivana Machackova,
and it may possess some auxin-like eects. Finally, it may act as a signal for Institute of Experimental Botany, Academy of
interaction of plants with herbivores and pests. Further research is needed to Sciences of the Czech Republic, Rozvojova
clarify these potential functions. 135, 165 02 Prague 6, Czech Republic.
E-mail: machackova@ueb.cas.cz
Received July 26, 2005;
accepted July 26, 2005.

solvents ethanol [14], 10% Na2CO3 [11], phosphate-


Introduction
buered saline [12], or potassium phosphate buer [15].
After its original discovery in the bovine pineal gland [1], However, Poeggeler and Hardeland [16] pointed out that
melatonin was found in many species of animals. Melatonin material from unicellular eukaryotes and plants often
is widespread and most extensively studied in vertebrates contains compounds (e.g. chelated iron or redox-active
mammals (e.g. [2, 3]), birds, reptiles, amphibians, bony biomolecules) that promote melatonin degradation via
shes, cartilagenous shes (reviewed in Ref. [4]), and photooxidation or free radical-mediated oxidation. They
cyclostomes [5]. However, its occurrence was also con- recommend 0.4 m HClO4 for protein precipitation (which
rmed in several invertebrate phyla (reviewed in Refs [68]). eliminates possible enzymatic melatonin degradation), and
The rst report of melatonin presence outside the animal potent hydroxyl radical scavengers (acetone, Tris/HCl, or
kingdom, in the dinoagellate alga Lingulodinium polye- Tricine/NaOH buer) with added antioxidants for extrac-
drum [9], suggested that it is a very ubiquitous compound, tion. The procedure should also be carried out in dim red
and that at least some of its functions might be evolutio- light or darkness to avoid photooxidation [9, 16]. Several
narily conserved. Since then, melatonin was indeed found in researchers subsequently used acetone-, Tris-, or Tricine-
many algae and in several species of fungi, protists, and based extraction mixtures, sometimes including antioxi-
even prokaryotes (reviewed in Refs [8, 10]). The work in dants, HClO4, or the chelator EDTA [9, 1721]. Hernan-
Lingulodinium also prompted a search for melatonin in dez-Ruiz et al. [22] also added an antioxidant (butylated
higher plants. The rst reports, published in 1995 [1113], hydroxytoluene) to their extraction solvent, ethyl acetate.
clearly demonstrated its presence in various angiosperm Melatonin was sometimes measured directly in the
species. After 10 yr of research, it is obvious that melatonin extracts [9, 12, 14, 20]. Nevertheless, Van Tassel and
is rather common in higher plants but there still remain ONeill [23] and Kolar [24] found further sample purica-
many unanswered questions. In this mini-review, we focus tion highly desirable because it removes compounds that
on two issues: (i) evaluation of methods employed for would otherwise interfere with melatonin determination.
sample preparation and melatonin determination in plants, Several studies used solid-phase extraction on C18 car-
(ii) hypotheses and evidence concerning melatonins func- tridges, sometimes followed by high-performance liquid
tions in plants. chromatography (HPLC) fractionation [17, 18, 21]. Other
authors extracted melatonin from an aqueous extraction
solution into an organic solvent chloroform or diethyl-
Methods of melatonin determination in
ether [11, 15]. Immunoanity chromatography was also
higher plants employed for sample purication [25, 26]. In this case,
Various methods of melatonin extraction, purication, and immunoanity columns were chosen because they were
determination were employed for samples from algae to successfully used before liquid chromatography mass
higher plants. Some authors used very simple extraction spectrometry (LC-MS) analysis of melatonin in human

333
Kolar and Machackova

serum [27]. Moreover, immunoanity purication is also deteriorated. The possibility of matrix eects should
part of some clean-up protocols for determination of plant therefore be kept in mind while developing methods for
hormones, e.g. cytokinins [28] and indole-3-acetic acid [29]. LC-MS measurement of melatonin in plants. Matrix
Melatonin was determined in plants by methods previ- eects, when encountered, can be minimized or eliminated
ously employed in animals. The methods dier in their by a more selective extraction, a more ecient chromato-
sensitivity and specicity. HPLC with uorescence detec- graphic separation, or a change of the ionization method
tion was not sensitive enough to prove melatonin presence [37, 38].
in C. rubrum shoots [24] but was successfully used to
quantify melatonin in Chinese medicinal herbs [30]. HPLC
with eletrochemical detection is more sensitive in compar-
Melatonin occurrence in higher plants
ison [16] and was often used in algae and higher plants (e.g. Melatonin was found in many species of angiosperms, in
[9, 14, 15, 20]) in spite of its relatively low specicity many both dicotyledons and monocotyledons. Its occurrence was
compounds with comparable oxidation potentials may have not yet investigated in other groups of higher plants
retention times very similar to melatonin. Radioimmuno- mosses, ferns, gymnosperms, etc. The rst preliminary
assay (RIA) is more specic and is frequently employed in report [13] was in P. nil (Japanese morning glory) where
animals (e.g. [3133]). However, detailed investigations melatonin was determined by RIA and GC-MS. Melatonin
suggest that plant extracts contain many substances cross- was then identied in Nicotiana tabacum and in edible
reacting with anti-melatonin antibodies [23]. Melatonin organs of tomato, banana, cucumber, and beetroot; it was
levels in plants determined by RIA alone and not validated not detected in potato. The levels were 2510 pg/g fresh
by other methods [11, 12] may thus be overestimated, as weight as determined by RIA, and melatonin identity in
was demonstrated, e.g. for Pharbitis nil and tomato [18]. In tomato extracts was conrmed by GC-MS spectra [11].
contrast to animals, melatonin RIA is therefore not a Hattori et al. [12] reported melatonin in 24 species of edible
reliable method in plants. Gas chromatography mass plants from 12 families of both dicotyledons and mono-
spectrometry (GC-MS) or LC-MS, which oer high sensi- cotyledons. Its content ranged from 10 to 5300 pg/g fresh
tivity and excellent detection specicity, can overcome the weight (RIA) and was generally the highest in seeds of
problems discussed above. Melatonin can be measured Poaceae, e.g. of rice and oat. Melatonin concentrations up
directly by LC-MS or it can be converted to a volatile (e.g. to 250 pg/g fresh weight were later detected in Chenopodium
pentauoropropionyl [34]) derivative and analyzed by rubrum shoots using LC-MS/MS [21]. Since then, the
GC-MS. Both methods have been used for melatonin presence of melatonin was reported in many other plants,
quantication in animals (e.g. [34, 35]). In plants, CG-MS with concentrations usually in the range of picograms to
was applied to conrm melatonin identity [11] and to nanograms per gram of tissue, as summarized in Table 1.
measure melatonin content in P. nil and tomato [18]. Liquid There is obviously convincing evidence that melatonin
chromatography tandem mass spectrometry (LC-MS/ occurs in higher plants. However, not all the data are
MS) was also used in several studies to prove melatonins equally reliable. Most importantly, some researchers did
identity (e.g. [19, 22, 30]) or to determine its levels [21, 25]. not prove melatonin identity beyond a reasonable doubt,
Liquid chromatography tandem mass spectrometry e.g. by mass spectrometric methods. In addition, some of
oers distinct advantages: rst, it does not require melato- the reported levels may be inaccurate. On one hand,
nin derivatization in contrast to GC-MS, and secondly, the recovery of extraction procedures was not properly deter-
double ion selection method provides the greatest specicity mined in some cases. Because melatonin is rather unstable
for melatonin currently achievable. However, Kolar [26] in plant extracts, the published concentrations might be
and J. Kolar and K. Wolf (unpublished data) observed that underestimated [6]. On the other hand, it was already
the area of melatonin peak detected by LC-MS/MS was mentioned above that plant material contains large
severely reduced in some series of samples. This was likely amounts of compounds that cross-react with antibodies
due to a so-called matrix eect. This phrase refers to used in RIA, as was pointed out by Van Tassel and ONeill
changes (usually reduction) in ionization eciency and [23]. When RIA was employed as the only method of
consequently in the peak area of an analyte, which are melatonin quantication (e.g. [11, 12]), the values may have
caused by coeluting compounds from biological material been overestimated. Indeed, several plants that were
and/or from chemicals or labware used for sample prepar- reported to have high melatonin levels, e.g. oat and ginger
ation. Matrix eects are often observed in LC-MS/MS [12], were later found to contain only trace amounts [17].
analyses [3638]. In the case discussed here, it would be Also, RIA measurements provided much higher melatonin
expected that the signal of the 2H-melatonin internal concentrations than GC-MS in P. nil and tomato [18]. As
standard in tissue extracts spiked with 2H-melatonin would regards the frequently used HPLC methods with electro-
be diminished in comparison with a solution containing chemical or uorescent detection, it would be desirable to
only the pure substance. This was indeed demonstrated for conrm the quantitative results (and not only melatonin
the aected plant samples (E. Witters, J. Malbeck and A. identity) by substantially more specic mass spectrometric
Travn ckova, unpublished results). The matrix eect does detection.
not pose a serious problem for melatonin quantication Melatonin daily proles have only been studied in two
because the ionization of both the endogenous compound species so far; both plants are important models for
and the deuterated internal standard added to each sample research on photoperiodic ower induction. Preliminary
should be inuenced to the same extent [39]. However, it is results suggested a diurnal rhythm with a night maximum
obvious that the detection limit of the method is greatly in P. nil shoots [13]. However, repeated experiments did not

334
Melatonin in higher plants

HPLC-ECD; validated by LC-MS/MS and RIA

HPLC-ECD; validated by LC-MS/MS and RIA

FW, fresh weight; DW, dry weight; HPLC, high-performance liquid chromatography; ECD, electrochemical detection; FD, uorescence detection; LC-MS/MS, liquid chromatography tandem
show any clear pattern of changes in a 12 hr light/12 hr
dark regime [18]. In contrast, a clear daily rhythm with a

HPLC-ECD; LC-MS/MS to conrm identity


HPLC-FD; LC-MS/MS to conrm identity
Method of melatonin determination
sharp maximum at night and very low levels during the day
was found in shoots of C. rubrum [21]. In this plant, the

RIA; HPLC-ECD to conrm identity


RIA; HPLC-FD to conrm identity
highest melatonin concentrations always occur 6 h before
RIA; GC-MS to conrm identity

lights on in dierent photoperiodic regimes [25].


Very little is known about melatonin biosynthesis in
higher plants. In one study, 14C-tryptophan was fed to in
vitro grown Hypericum perforatum plantlets. Tryptophan
LC-MS/MS, RIA was metabolized to indoleacetic acid, tryptamine,
5-hydroxytryptophan, serotonin, and melatonin [19].

GC-MS, RIA
not reported 5-Hydroxytryptophan and serotonin are melatonin pre-
HPLC-ECD

LC-MS/MS
cursors in vertebrates [40], suggesting the melatonin
biosynthetic pathway in higher plants might be similar to
that in animals.

Possible melatonin functions in higher


up to 3800 ng/g DW (more than 10 ng/g DW in 64 species)

plants
edible plants: 2510 pg/g FW; tobacco: 40100 pg/g FW

Regulation of circadian rhythms and photoperiodic


reactions
Most vertebrates studied so far exhibit daily rhythms in
morning glory: up to 12 pg/g FW (GC-MS);
Melatonin concentration

blood levels of melatonin, with low concentrations during


the day and high at night. These rhythms persist under
tomato: up to 17 pg/g FW (GC-MS)

constant darkness and are therefore circadian [4, 41]. It was


up to 250 pg/g FW (LC-MS/MS)

thus proposed that melatonin mediates time-of-day infor-


mation to target cells throughout the body (e.g. [41]).
Several lines of evidence indeed support the involvement of
up to 4000 nmol/g FW

melatonin in vertebrate circadian rhythmicity. Importantly,


944000 nmol/g DW
2190 ng/g dry seed

circadian rhythms are aected by exogenous melatonin


up to 100 pg/g FW

mass spectrometry; GC-MS, gas chromatography mass spectrometry; RIA, radioimmunoassay.


105300 pg/g FW

0.12.5 lg/g DW

treatment (reviewed in Refs [42, 43]): depending on the


213 ng/g FW

928 ng/g FW

experimental setting, melatonin can cause arrhythmicity,


change the free-running period or phase, or entrain free-
Table 1. A summary of important publications that reported melatonin in higher plants

running rhythms. Daily melatonin proles may also encode


photoperiodic information as lengthening of the night
prolongs the duration of elevated melatonin levels [4, 41].
Melatonin clearly aects mammalian photoperiodism
St. Johns wort (Hypericum perforatum), developing owers

(reviewed in Refs [41, 44, 45]). Treatments that disrupt


24 edible plants (monocot and dicot species), edible parts

feverfew (Tanacetum parthenium), Hypericum perforatum,

the daily oscillations in melatonin levels (removal of the


huang-qin (Scutellaria baicalensis), in vitro propagated

pineal gland or continuous-release melatonin implants)


morning glory (Pharbitis nil), shoots; tomato, fruit

inuence photoperiodic reactions. Photoperiodic responses


Chinese medicinal herbs (108 species screened),
Plant species and organs analyzed

and two Scutellaria species, leaves or owers

are mainly determined by the daily duration of high


melatonin levels; for example, melatonin application at
shoots from 26 distinct germplasm lines

appropriate times of day can elicit short-day responses in


5 edible species, edible parts; tobacco

animals kept in long days. The situation is more complex in


tart cherries (Prunus cerasus), fruit

nonmammalian vertebrates where pinealectomy and/or


15 species of edible plants, seeds
lupin (Lupinus albus), hypocotyl

melatonin administration inuence seasonal reproduction


Chenopodium rubrum, shoots

Chenopodium rubrum, shoots


(Nicotiana tabacum), leaves

in some species but not in others [4]. In vertebrates,


melatonin thus regulates circadian rhythms and photoperi-
odism. Daily and/or circadian rhythms in melatonin levels
were also found in some invertebrate species, suggesting
various organs

similar roles in rhythmicity and/or photoperiodism


(reviewed in Ref. [6, 7]).
Melatonin was also found in many species of algae
(reviewed in Ref. [10]). Five species of green algae
Chlamydomonas, Dunaliella, and Acetabularia show daily
melatonin rhythms with night maxima which continue in
Reference

constant conditions [46]. There is also some evidence for


similar diurnal oscillations of melatonin levels in the
[104]
[105]

[106]
[15]
[11]

[12]
[22]
[30]

[21]
[14]

[18]

[25]

red alga Porphyra umbilicalis [47]. In the dinoagellate

335
Kolar and Machackova

L. polyedrum, melatonin concentrations also exhibit a daily the short-day C. rubrum and the long-day A. thaliana, this
rhythm with a night maximum [9]. In addition, melatonin eect could be a more general phenomenon among higher
and its metabolite 5-methoxytryptamine promote photope- plants. The lack of melatonins inuence on owering in
riodically controlled encystment [48] and aect the circa- some species (see above) might have been caused merely by
dian bioluminescence rhythm [6] in this organism. It was an inappropriate experimental setup or by an inappropriate
also reported that melatonin application causes phase shifts method or timing of treatment. Importantly, only the
of a bioluminescence rhythm in the dinoagellate alga ability of melatonin to promote owering in noninductive
Pyrocystis acuta [49] and possibly in several other dino- photoperiods was tested in most cases. Nevertheless, it
agellates [10]. These ndings resemble melatonins roles in should be stressed that melatonin concentrations required
animal rhythmicity and photoperiodism, suggesting that for the inhibition of owering exceed the naturally occur-
the involvement of melatonin in time measurement might ring levels by several orders of magnitude, at least in
be evolutionarily conserved and shared by most eukaryotes. C. rubrum where the endogenous concentrations were
Several authors therefore tested whether melatonin found to be 10)12 mol/g fresh weight or less [21]. The
aects circadian rhythms and/or photoperiodic reactions results are thus encouraging but do not prove that
also in higher plants. This possibility is indirectly supported endogenous melatonin aects plant photoperiodic
by the nding that melatonin levels in the dicot plant responses. It is of course possible that only a small
C. rubrum exhibit a diurnal rhythm with a night maximum proportion of the applied melatonin is transported to target
[21, 25]. Because melatonin application in long days causes cells. But it cannot be ruled out that high levels of exogenous
short-day responses in some vertebrates, eects of melato- melatonin may in fact suppress owering by a mechanism
nin administration were investigated in several species of not related to the mode of action of the endogenous
plants with photoperiodically controlled owering. How- compound. Further studies are needed to identify the exact
ever, no promotion of owering after melatonin application mechanism(s) of melatonins action on owering.
was found in the short-day plant P. nil [17]. Similarly, Very little is known about melatonin eects on circadian
melatonin treatment 1, 2, or 4 hr before lights o does not rhythms of higher plants. Kolar [26] tested melatonin,
induce owering of the water plants Spirodela polyrhiza, several related indole derivatives, and compounds that are
Lemna minor, and Lemna trisulca in noninductive photo- melatonin agonists or inhibitors of its biosynthesis in
periods [50]. 10)4 or 5 10)4 m melatonin also does not animals. Neither melatonin nor any of the other chemicals
signicantly inuence owering of the long-day plant specically aected two circadian rhythms: cotyledon
Chenopodium murale [51]. movement in C. rubrum (after pulse application at two
The most convincing eect of melatonin on a photope- dierent circadian times), and bioluminescence in
riodic response in a higher plant was found in the short-day N. tabacum seedlings carrying a luciferase reporter gene
plant C. rubrum [52]. In contrast to some vertebrates, controlled by a circadian clock-regulated cab2 promoter
melatonin application does not induce a short-day response (during continuous application to nutrient medium). Mela-
(i.e. owering) after a short (68 hr) noninductive night. tonin also does not inuence the circadian rhythm of
Instead, 100 or 500 lm melatonin applied 1 hr before the photoperiodic time measurement in C. rubrum (see above).
beginning of an inductive 12-hr darkness signicantly These data indicate that melatonin probably does not
lowers the percentage of owering plants. Uptake experi- regulate daily rhythms in higher plants; but some eects
ments revealed that high melatonin levels in the plant (50 might be perhaps discovered in the future if other treatment
90% of the applied amount) are reached during the rst schedules, species, or rhythms are investigated.
half of darkness and are maintained until at least 24 hr
after the end of this dark period. Exogenous melatonin
Other functions related to those in animals
could therefore inuence both photoperiodic ower induc-
tion and/or subsequent developmental processes in the In animals, melatonin not only regulates photoperiodism
shoot apical meristem. Because 500 lm melatonin inhibits and circadian rhythmicity but also has many other func-
owering only when applied 3 hr before or during the rst tions. Melatonin may play some of these roles also in higher
half of a 12-hr dark period, it probably aects some early plants.
steps of the transition to owering. The rst step is the Melatonin binds to Ca2+-calmodulin in animal cells [54]
measurement of night length by a circadian oscillator. and antagonizes some of its eects [55]. In MDCK cells
Chenopodium rubrum has a circadian rhythm in photope- treated with 10)9 m melatonin, melatonin co-localizes with
riodic sensitivity to a single night of various durations [53]. calmodulin and changes its subcellular distribution [56].
However, melatonin treatment has no eect on the period Because Ca2+-calmodulin regulates many calcium-depend-
or phase of this rhythm. Exogenous melatonin must thus ent cellular functions, these ndings suggest that melatonin
control some other early process(es) of C. rubrum transition may aect a broad range of processes via this interaction.
to owering. For example, melatonin inhibits the activity of Ca2+-
Melatonin application also delays owering of the long- calmodulin-dependent nitric oxide synthase in rat cerebel-
day plant Arabidopsis thaliana [26]. Arabidopsis thaliana lum, possibly by acting as a calmodulin antagonist [57]. On
plants treated with 100 or 500 lm melatonin ower slightly the contrary, melatonin showed no calmodulin antagonism
later than control plants, especially in terms of their in the case of T-lymphocyte activation [58]. Calmodulin is a
ontogenetic age, i.e. leaf number; numbers of rosette and calcium signal transducer also in plants [59], so melatonin
total leaves at anthesis are signicantly increased by eects on plant processes controlled by calmodulin are
810%. Because melatonin suppresses owering in both worth investigating.

336
Melatonin in higher plants

Melatonin aects the cytoskeleton, causing either micro- originating in mitochondria) and putative existence of
tubule enlargement or depolymerization. It was proposed mitochondrial binding site for melatonin [84, 85]. In in vitro
that at physiological concentrations (10)9 m) melatonin experiments, melatonin prevented oxidative damage in
antagonizes the inhibition of tubulin polymerization caused mitochondria induced by t-butyl hydroperoxide [86]. Mela-
by calmodulin and thus stimulates microtubule growth. At tonin also elevated ATP production in mitochondria [87].
pharmacological levels (10)710)5 m), melatonin binds to The proposed actions of melatonin at the mitochondrial
tubulin and prevents its polymerization (reviewed in Ref. level have recently been reviewed by Leon et al. [88].
[60]). These eects were observed in both animals and Melatonin shows a synergism with classical antioxidants
plants. In higher plants, 10)4 m melatonin increases biref- like GSH and vitamins E and C [89]. Under high oxidative
ringence of the mitotic spindle (i.e. stimulates microtubule stress melatonin was shown to be superior to vitamins E
assembly) in Haemanthus katherinae [61]. In contrast, and C in reducing damage [65]. Melatonin acts as an ABTS
8 10)4 m melatonin causes microtubule depolymerization cation radical scavenger with much higher eciency than
and therefore disrupts the mitotic apparatus in onion root other antioxidants with which melatonin acts synergistically
tips [62]. [67].
Finally, melatonin is a radical scavenger and an anti- In plants, antioxidant properties of melatonin were
oxidant in animals. Melatonin was shown to protect described as well, but the functioning of melatonin as an
organisms against reactive oxygen and nitrogen species antioxidant in vivo is much less clear than in animals. In the
(ROS and RNS) [6366]. Melatonin is in many animals alga L. polyedrum, high levels of melatonin rescued cells
produced also in other organs than pineal gland and a from lethal oxidative stress caused by H2O2 and this eect
hypothesis was suggested that this melatonin has other than was not because of induction of antioxidative enzymes [90].
hormonal functions, e.g. radical scavenging [67]. Actions of Sublethal oxidative stress induced by dierent agents was
melatonin as an antioxidant can be classied into four also counteracted by melatonin [8, 91]. In tomato, a
categories: (a) direct free radical scavenging; (b) stimulation correlation between susceptibility to ozone and melatonin
of activities of antioxidant enzymes; (c) increasing the levels was described [11]. Melatonin was also reported to
eciency of mitochondrial oxidative phosphorylation and delay senescence of leaves, both in monocots and dicots
reducing electron leakage; (d) augmenting the eciency of [92]. High levels of melatonin found in seeds led to the
other antioxidants [64]. suggestion that melatonin functions as a protective anti-
Melatonin has the ability to neutralize the highly toxic oxidant [14].
hydroxyl radical (OH) [68]. The proposed product of this
reaction is cyclic 3-hydroxymelatonin, which also has
Functions specific to higher plants
antioxidant properties. Reaction of melatonin with hydro-
gen peroxide was shown in vitro and its product again has Not all melatonins functions in higher plants have to
antioxidant properties [69, 70]. These cascades of scaven- duplicate those in animals. For example, melatonin (N-
ging events may be the reason for the unexpectedly high acetyl-5-methoxytryptamine) is structurally related to the
eciency of melatonin in reducing radical damage in vivo. plant hormone auxin (IAA, indole-3-acetic acid); both are
Melatonin was shown also to scavenge superoxide (O2 ) indole derivatives. It is therefore possible that melatonin
and NO radicals [7173] and also haloperoxyradicals, e.g. (either endogenously occurring or exogenously applied)
CCl3O2 [74]. may exhibit some auxin-like eects in plants. It could
Melatonin increases activities of several antioxidant hypothetically bind to auxin receptors and act directly as an
enzymes: superoxide dismutase (SOD, both MnSOD and auxin agonist. However, it does not meet the requirements
CuSOD), catalase, glutathione peroxidase (GPx), glutathi- for an agonist very well. It was concluded from a
one reductase (GRd) and glucose-6-phosphate dehydroge- comparison of auxin activities of various compounds that
nase [63, 75]. Melatonin stimulates the rate-limiting enzyme the minimal structural requirements for auxin activity are
of glutathione (GSH) synthesis, c-glutamylcysteine syn- the presence of an aromatic group and a carboxyl group or
thase, and thus increases intracellular concentration of groups that can mimic them in terms of electron distribu-
GSH [76]. Moreover, amplication of GPx activity in rat tion and polarity [93]. The melatonin molecule does have an
brain by melatonin was observed and melatonin deciency aromatic (indole) part but lacks the acidic carboxyl or
after pinealectomy reduces the activity of this enzyme [77]. similar group.
Melatonin also increases the expression of genes coding for In agreement with these theoretical considerations, it was
antioxidative enzymes, especially SOD [78]. Additionally, demonstrated that melatonin has no auxin activity in two
melatonin has antiapoptotic eects [79] and is able to model systems: the wheat coleoptile segment straight
prevent neurons in patients with Alzheimer disease from growth biotest [26] and the decapitated Coleus shoot
mutilation and death [80, 81]. Similar benecial eects of phototropic test [94]. But a recent paper by Hernandez-
melatonin have been observed in prematurely born children Ruiz et al. [22] shows an auxin-like activity of melatonin in
with septic shock [82] and in respiratory distress syndrome Lupinus albus. Applied melatonin stimulated elongation
[83]. growth of L. albus hypocotyls in a concentration range
The specic functions of melatonin at the mitochondrial similar to the promotive concentrations of IAA. In three
level are under intensive investigation. A relationship is dierent biotests, maximal growth activation by melatonin
suggested on the basis of high ROS and RNS production in was 2263% of that caused by IAA treatment. Moreover,
mitochondria, occurrence of GPx and GRd in mitochon- the highest levels of endogenous IAA and melatonin were
dria, anti-apoptotic eects of melatonin (apoptotic signals detected in the hypocotyl zone with the maximal growth

337
Kolar and Machackova

rate, supporting the notion that melatonin may indeed have [8]. We should also stay open to the possibility that in
a hormonal function in this case. plants, melatonin can have function(s) dierent from those
Although these results are very interesting, future studies in animals. For example, it may possess some auxin-like
should also consider the possibility that the applied eects [22], or act as a signal in communication with
melatonin could be metabolized to IAA or an IAA agonist herbivores or pests. Only further research will dene the
in plant tissues. For instance, melatonin can be converted roles of melatonin in plants.
to 5-methoxyindoleacetic acid, at least in animals [95], and In the last several years, attention was also paid to
this compound exhibits low auxin activity in the split pea possible use of plants with high melatonin levels as a
stem bioassay [93]. dietary supplement to increase its blood plasma levels in
Murch et al. [96] studied melatonins eects on organo- humans (e.g. [97]). Plant melatonin may indeed improve
genesis in in vitro cultured stem sections of H. perforatum. human health. As described above, melatonin is a potent
They tested melatonin, serotonin, the auxin IAA, and several radical scavenger and antioxidant. Melatonin seems to
inhibitors of auxin or indoleamine action or transport. In act protectively in some diseases, e.g. in neurodegenera-
most variants supplemented with the inhibitors (alone or tive diseases [102]. Dietary intake of melatonin has been
combined with IAA), reduced root regeneration correlated shown to increase melatonin uptake by tumor tissue and
with decreases in the levels of both IAA and melatonin. While retard tumor growth [103]. Some medicinal plants, e.g.
IAA alone strongly stimulated root formation, melatonin H. perforatum, contain high melatonin levels (see Table 1
addition to the culture medium had only minimal inuence for references). Given the current strong demand for
on root regeneration. Further experiments are therefore natural healthcare products, this line of research can
necessary before we can assess the role of melatonin in plant bring promising and perhaps commercially interesting
organogenesis, either in vitro or in vivo. results.
Melatonin may also have ecological functions in higher
plants. Reiter et al. [97] suggested that melatonin in plants
References
could increase the levels of this indoleamine in animals that
consume such plants. Indeed, this group recently found 1. Lerner AB, Chase JD, Takahashi Y et al. Isolation of
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