Download Melatonin stimulates the expansion of etiolated lupin cotyledons

Plant Growth Regul (2008) 55:29–34
DOI 10.1007/s10725-008-9254-y
ORIGINAL PAPER
Melatonin stimulates the expansion of etiolated lupin
cotyledons
Josefa Hernández-Ruiz Æ Marino B. Arnao
Received: 17 October 2007 / Accepted: 2 January 2008 / Published online: 11 January 2008
Ó Springer Science+Business Media B.V. 2008
Abstract Melatonin (N-acetyl-5-methoxytryptamine)
is an indoleamine which is structurally related to
tryptophan, serotonin and indole-3-acetic acid (IAA),
among other important substances. Many studies have
clearly demonstrated its presence in different plant
organs, including roots, stems, leaves, flowers, fruits
and seeds. Since it discovery in plants in 1995, authors
have postulated many physiological roles for melatonin, although research into this molecule in plants is
still in its infancy. The data presented in this study
demonstrate that melatonin stimulates the expansion of
etiolated cotyledons of lupin (Lupinus albus L.) to a
similar extent to that observed for IAA but less than in
the case of kinetin. Endogenous melatonin in imbibed
cotyledons has been quantified using a liquid chromatography method with fluorescence detection and
capacity of cotyledons to absorb melatonin has been
determined. The observed effect of melatonin on lupin
cotyledon expansion can be added to the other effects
demonstrated by our group such as its role as growth
promoter and rooting promotor in adventitious and
lateral roots.
Keywords Auxin Cotyledon IAA Melatonin Growth Lupinus
J. Hernández-Ruiz M. B. Arnao (&)
Department of Plant Physiology, Faculty of Biology,
University of Murcia, 30100 Murcia, Spain
e-mail: [email protected]
Introduction
In vertebrates, melatonin (N-acetyl-5-methoxytryptamine; Fig. 1), a well known animal hormone, is
released by the pineal gland although it is formed
in numerous other organs. As a hormone, it plays a
key role in various physiological processes such as
circadian rhythmicity, sleep and seasonal photoperiod regulation, sexual and reproductive behavior
and immunological enhancement (Foulkes et al.
1997; Reiter 1993). Melatonin not only acts as a
hormone, but has also been studied for its role in
scavenging different types of reactive oxygen and
nitrogen species (Reiter et al. 2001). Besides being
present in vertebrates, this molecule has also been
detected in other organisms, including insects,
unicellular organisms, algae and bacteria (Hardeland and Fuhrberg 1996; Hardeland and Poeggeler
2003).
Since 1995, melatonin has also been known to
exist in the roots, leaves, fruits and seeds of a
considerable number of plant species (Dubbels et al.
1995; Kolar et al. 1995; Manchester et al. 2000). In
most cases, its study in plants has mainly focused on
the quantification of melatonin levels in different
plant organs and species. However, very little is
known concerning the physiological role played by
melatonin in plants (Kolar and Machackova 2005;
Arnao and Hernández-Ruiz 2006), although inconclusive attempts have been made to seek a role for
this indolic compound as a photoperiodic and
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circadian regulator (van Tassel et al. 2001; Kolar
et al. 1997; Wolf et al. 2001). The effect that
exogenous melatonin only affects the flowering of
the short-day plant Chenopodium rubrum at high
doses (0.5 mM), particularly during the early stages
of photoperiodic induction, goes against it being
considered as a physiological agent (Kolar et al.
2003). However, the biosynthetic route of melatonin
and serotonin from tryptophan has been partially
characterised in cultured St. John‘s wort (Hypericum
perforatum L.) cells (Murch et al. 2000; 2002).
Recently, we mentioned for the first time a
possible action for melatonin in plants, whereby
indoleamine promotes vegetative growth in etiolated lupin (Lupinus albus L.) hypocotyls in a
similar manner to indole-3-acetic acid (IAA; Fig. 1)
(Hernández-Ruiz et al. 2004). Melatonin and IAA
were also seen to be distributed in lupin tissues in a
similar concentration gradient. We also confirmed
that melatonin acts as a growth promoter in
coleoptiles of wheat, barley, canarygrass and oat,
presenting activity with respect to IAA of between
10 and 55%. Of particular note was the inhibitory
growth effect that melatonin presented on the
monocot roots assayed, which was similar to that
exercised by IAA (Hernández-Ruiz et al. 2005). We
also described the effect of melatonin on the
regeneration of lateral and adventitious roots in
etiolated hypocotyls of Lupinus albus, comparing
the effect of different concentrations of melatonin
and IAA on root promotion (Arnao and HernándezRuiz 2007a).
In this paper, we describe the positive effect of
melatonin on lupin cotyledon expansion. Also, a
quantitative comparison of this effect with kinetin
and IAA is presented. Liquid chromatography with
fluorescence detection is used to measure endogenous
melatonin in cotyledons and also to estimate exogenous melatonin absorption by cotyledons during the
imbibition process.
Fig. 1 Chemical structures
of melatonin (N-acetyl-5methoxytryptamine) and
indole-3-acetic acid (IAA)
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Plant Growth Regul (2008) 55:29–34
Materials and methods
Plant material
Seeds of lupin (Lupinus albus L.) were sterilized in
10% hypochlorous solution for 5 min and soaked in
distilled water for 24 h at 24°C in darkness.
Reagents
The indolic compounds, IAA, 5-methoxyindole3-acetic acid (5MIAA) and melatonin (N-acetyl-5methoxytryptamine), were purchased from Acros
Organics Co. (Geel, Belgium). Kinetin was obtained
from Sigma (Madrid, Spain). The solvents ethyl
acetate, water and acetonitrile (HPLC grade) were
obtained from Scharlau Chemie (Barcelona, Spain).
The different reagents and salts (analytical grade)
used to prepare the incubation solutions were
obtained from Merck (Darmstadt, Germany).
Induction of growth
Fully imbibed etiolated cotyledons, without the
embryo, were placed in Petri dishes containing
medium (10 mM potassium-phosphate buffer, pH
6.2) with 0.1, 1, and 10 lM concentrations of kinetin,
IAA, melatonin or 5MIAA. A medium consisting only
of 10 mM potassium-phosphate buffer (pH 6.2) was
used as control. IAA, melatonin and 5MIAA were
prepared in pure ethanol and dissolved to final
concentrations in buffer, being the concentration of
ethanol less than 0.1%. The area and fresh and dry
weight of the cotyledons in each treatment were
registered initially and after 48 h incubation at 24°C in
darkness. Between 8–12 cotyledons were used in each
treatment, which was repeated 8 times. The increase in
area of the cotyledons was measured using the software
application Adobe Photoshop 6.0 for Windows.
Plant Growth Regul (2008) 55:29–34
The capacity of the cotyledons to incorporate
exogenous melatonin was made by incubating dry
sterilized lupin seeds in media (10 mM potassiumphosphate buffer, pH 6.2) containing the melatonin
concentrations of 1, 10 nM, 0.1 and 1 lM, for 24 h,
at 24°C in darkness. The melatonin content in
cotyledons was determined by HPLC immediately.
Indole analysis
To measure the melatonin content in water-imbibed
cotyledons, the extraction and chromatographic method
previously developed for lupin and monocots was used
(Hernández-Ruiz et al. 2004; 2005). Briefly, etiolated
cotyledons (2–3 g) were cut into sections (3–5 mm)
and, without homogenization, were placed in vials
containing 15 ml ethyl acetate with butylated hydroxytoluene (5 lM), for 15 h at 4°C in darkness with
shaking. The sections were then discarded and the
solvent evaporated under vacuum using a SpeedVac
ThermoSavant mod. SPD111V coupled to a refrigerated
vapor trap mod. RVT400 (USA). The dry residue was
redissolved in 1 ml of acetonitrile, filtered (0.2 lm) and
analysed by HPLC with fluorescence detection. The
percentage of recovery using exogenous standard
melatonin added to cotyledon samples was estimated,
the losses in our method being about 4–6%, which is in
accordance with our previously published data.
HPLC (Jasco Co., Tokyo, Japan) using an RP-C18
column with fluorescence detection (Jasco, model
FP-2020-Plus) was used to measure melatonin levels
in the cotyledons. An excitation wavelength of
280 nm and an emission wavelength of 348 nm were
used. The isocratic mobile phase consisted of water:
acetonitrile (75:25) at a flow rate of 0.5 ml/min. An
on-line fluorescence spectral analysis (using the Jasco
FP-2020 fluorescence detector), comparing the excitation and emission spectra of standard melatonin with
the corresponding peak in the samples was applied.
Identification was confirmed by tandem mass spectrometry (MS/ESI+), as previously described (HernándezRuiz et al. 2004).
Statistical analysis
For the bioassay data and indole quantitation data,
differences were determined using the SPSS 10
31
program (SPSS Inc., Chicago) applying the LSD
multiple range test to establish significant differences
at P \ 0.05.
Results and discussion
The typical cotyledon expansion assay described by
Letham (1971) in radish was used to characterize the
possible effect of melatonin on lupin. This bioassay
permits the effect of different compounds on the
growing capacity of cotyledon to be detected and
measured. However, only some species, mainly of the
leguminosae family, can be used in this bioassay. To
obtain precise measurements of overall increases in
cotyledon area the use of a digital-imaging tool is
recommended.
Figure 2a shows the effect of kinetin, IAA,
melatonin and 5MIAA on cotyledon area expansion
after 48 h of incubation at 24°C in darkness. The data
show an increase in area with respect to the control
incubation (only buffer medium) for all treatments,
except for 5MIAA. At 10 lM, kinetin induced the
highest response, there were no significant differences between kinetin, IAA and melatonin at 1 and
0.1 lM. In general, treatment with melatonin and
IAA increased the size of lupin cotyledons with
respect to the control, with only very slight differences between them. The treatment with 5MIAA,
a catabolite of melatonin detected in certain organisms
such as the dinoflagellate Lingulodinium (Hardeland
et al. 2007), did not lead to increase in area in lupin
cotyledons. The same behaviour was observed when
changes in the fresh weight of the cotyledons were
measured (Fig. 2b), three of the agents, kinetin, IAA
and melatonin, clearly affecting growth, while
5MIAA did not. Again, while 10 lM kinetin caused
a significant fresh weight increase, IAA and melatonin had a lower effect with minor differences
between them.
In contrast, the dry weight measurements in
control cotyledons (only imbibed with buffer) and
for each treatment (kinetin, IAA, melatonin and
5MIAA) showed no significant differences (Table 1),
indicating that the growth of cotyledons was due,
mainly, to increasing cell volume through cell
expansion, not as a result of cell division. In the
typical radish cotyledon bioassay using cytokinins,
structural changes are observed within the cotyledon
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Fig. 2 Effect of different concentrations of kinetin, IAA,
melatonin and 5MIAA on etiolated lupin cotyledon area (a)
and fresh weight (b). Data represent the mean values in each
treatment (0.1, 1.0 and 10 lM). Error bars represent standard
errors of the mean (n = 8). The different superscript letters
represent statistically significant differences at P \ 0.05
cells during expansion. Although a slight increase in
cell numbers is observed, the expansion of cotyledons
is mainly due to expansion of the pre-existing cells
and those cells formed by cell division (Thomas and
Katterman 1992). In the case of IAA and melatonin,
lupin cotyledon expansion was less pronounced than
with kinetin, probably because only cell expansion
occurs with both indolic compounds.
One important aspect of this effect of melatonin on
plant tissues is its possible action through acidification of the media. As we previously described
(Hernández-Ruiz et al. 2005), melatonin does not
induce acidification of the solutions after 24 h
incubations at different concentrations. The presence
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Plant Growth Regul (2008) 55:29–34
of barley coleoptile sections together with melatonin
causes a slight acidification of the media, which is
related to the growth-promotion of coleoptiles by
melatonin, similar to that of IAA, suggesting possible
ATPase-mediated activation. In the case of lupin
cotyledons, a similar effect was observed using
melatonin and IAA (data not shown).
The presence of melatonin has been described in
different lupin organs (hypocotyl, root) (HernándezRuiz et al. 2004; Arnao and Hernández-Ruiz 2007a).
In the present paper, we have analyzed the level of
endogenous melatonin in untreated imbibed cotyledons, applying an extraction and determination
method which used extraction without homogenization and quantitation by HPLC with fluorescence
detection. When endogenous melatonin was quantified in cotyledons (Table 2), its level was lower than
in other organs of etiolated lupin. We previously
demonstrated how melatonin appeared in 6-day-old
lupin hypocotyls, in which it was distributed in a
gradient form, in a similar way to IAA, the apical
tissues (next to the meristematic zone) showing the
highest levels (Hernández-Ruiz et al. 2004). Possibly,
melatonin is synthetized in the meristematic zone
(like IAA) and is transported basipetally through the
hypocotyl and also to the roots, but in a lesser amount
to the cotyledons. Nevertheless, to test whether
melatonin is really the agent that causes cotyledon
expansion, we imbibed dry cotyledons in media with
different melatonin concentrations. Table 2 shown
the level of melatonin measured in the cotyledons
after the imbibition process. The amount of melatonin
accumulated in the cotyledons increased with the
concentration of melatonin in the incubation medium.
Thus, at 1 nM practically the same amount of
melatonin as in the control was measured. In the
medium with 1 lM melatonin, a level of 81.2 ng
melatonin g FW-1 was estimated, a 63.44-fold
increase with respect to endogenous melatonin (control). These data show than melatonin was clearly
absorbed by the cotyledons, where it played some
role in the cellular expansion.
Many data point to a possible role for melatonin in
plants, as seen from the growth-stimulating effect
observed in lupin hypocotyls and monocot coleoptiles,
the growth-inhibiting effect observed in roots and the
in vivo organogenic effect which induces new adventitious and lateral roots in lupin (Hernández-Ruiz et al.
2004, 2005; Arnao and Hernández-Ruiz 2007a) and the
Plant Growth Regul (2008) 55:29–34
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Table 1 Dry weight of lupin cotyledon after 24 h of incubation, in darkness, with kinetin, IAA, melatonin and 5MIAA at different
concentrations
Incubation
medium
Control Kinetin Kinetin Kinetin IAA
IAA
(buffer) 10 lM 1 lM 0.1 lM 10 lM 1 lM
IAA
MEL MEL
0.1 lM 10 lM 1 lM
MEL
5MIAA 5MIAA 5MIAA
0.1 lM 10 lM 1 lM
0.1 lM
Dry weight,
mg
94.7
94.7
102.3
96.2
(±3.9)
(±4.6) (±6.7) (±4.4) (±5.8) (±4.6) (±4.2) (±3.4) (±3.3) (±4.2) (±3.0)
92.1
103.9
105.0
97.5
105.5
105.9
96.4
99.4
94.0
(±4.1)
(±4.4)
(± SE)
Table 2 Measurements of endogenous melatonin in cotyledons imbibed in 10 mM potassium phosphate, for 24 h in
darkness (Control) and in the same buffer with different melatonin concentrations
[Melatonin]
in imbibition
Melatonin
(ng g FW-1)
Multiplied
factor (9times)
Zero (Control)
1.28 ± 0.06
–
10-9 M
1.32 ± 0.07
91.03
10-8 M
2.47 ± 0.11
91.93
10-7 M
5.84 ± 0.25
94.56
10-6 M
81.21 ± 4.56
963.44
effect on cell expansion in lupin cotyledons described
in this paper. Taken together with other relevant data,
such as the in vitro organogenesis observed in Hypericum perforatum L. (Murch et al. 2000) and the
inhibition of ACC oxidase activity in lupin by melatonin and IAA (Arnao and Hernández-Ruiz 2007b), we
indicated that there is a great similarity with IAA in
some physiological responses. So, the co-presence of
melatonin and IAA in different plant tissues and
species supports the hypothesis that there is possible
co-participation in some physiological actions. However, the absence of a carboxylic group in the side chain
of melatonin is a serious handicap to acts on auxin
receptors, although the presence of specific melatonin
receptors in plant cells cannot be discarded. Nevertheless, more specific studies such as those previously
suggested (Arnao and Hernández-Ruiz 2006) are
necessary to establish the exact role of melatonin in
plants. Special attention should be focused on melatonin metabolism in plants (the possible biosynthetic
routes and the catabolism). A particularly important
aspect to study is the possible role of the metabolite
5-methoxy-indole-3-acetic acid, as suggested in the
interesting review recently presented by Hardeland
et al. (2007). So, the possible physiological role of
melatonin in plants, with the currently available data,
should be described very carefully, with attention to the
possible emergence and actuation of melatonin metabolites with auxin activity. As suggested in a recent note
(Arnao and Hernández-Ruiz 2007c) much more studies, with a diversity of focuses is necessary to provide a
solid data set concerning the role of melatonin in
plants.
Acknowledgments This work was supported by project
MCyT-BFU2006–00671 (co-financed by FEDER) and by
project 502/PI/04 from Fundación SENECA (C.A.R.Murcia).
J.H.R. has a contract with the University of Murcia.
References
Arnao MB, Hernández-Ruiz J (2006) The physiological function of melatonin in plants. Plant Signal Behav 1:89–95
Arnao MB, Hernández-Ruiz J (2007a) Melatonin promotes
adventitious and lateral root regeneration in etiolated
hypocotyls of Lupinus albus L. J Pineal Res 42:
147–152
Arnao MB, Hernández-Ruiz J (2007b) Inhibition of ACC
oxidase activity by melatonin and IAA in etiolated lupin
hypocotyls. In: Ramina A, Chang C, Giovannoni J, Klee
H, Perata P, Woltering E (eds) Advances in plant ethylene
research, Dordrecht, pp 101–103
Arnao MB, Hernández-Ruiz J (2007c) Melatonin in plants:
more studies are necessary. Plant Signal Behav 2:381–382
Dubbels R, Reiter RJ, Klenke E, Goebel A, Schnakenberg E,
Ehlers C, Schiwara HW, Schloot W (1995) Melatonin in
edible plants identified by radioimmunoassay and high
performance liquid chromatography-mass spectrometry.
J Pineal Res 18:28–31
Foulkes NS, Borjigin J, Snyder SH, Sassone-Corsi P (1997)
Rhythmic transcription: the molecular basis of circadian
melatonin synthesis. Trends Neurosci 20:487–492
Hardeland R, Fuhrberg B (1996) Ubiquitous melatonin: presence and effects in unicells, plants and animals. Trends
Comp Biochem Physiol 2:25–45
Hardeland R, Poeggeler B (2003) Non-vertebrate melatonin.
J Pineal Res 34:233–241
Hardeland R, Pandi-Perumal SR, Poeggeler B (2007) Melatonin in plants. Focus on a vertebrate night hormone with
cytoprotective properties. Funct Plant Sci Biotechnol
1:32–45
123
34
Hernández-Ruiz J, Cano A, Arnao MB (2004) Melatonin:
a growth-stimulating compound present in lupin tissues.
Planta 220:140–144
Hernández-Ruiz J, Cano A, Arnao MB (2005) Melatonin acts
as a growth-stimulating compound in some monocot
species. J Pineal Res 39:137–142
Kolar J, Machackova I (2005) Melatonin in higher plants.
Occurrence and possible functions. J Pineal Res 39:333–341
Kolar J, Machackova I, Illnerova H, Prinsen E, van Dongen W,
van Onckelen HA (1995) Melatonin in higher plant
determined by radioimmunoassay and high performance
liquid chromatography-mass spectrometry-mass spectrometry. Biol Rhythm Res 26:406–409
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 44:1407–1413
Kolar J, Johnson CH, Machackova I (2003) Exogenously
applied melatonin affects flowering of the short-day plant
Chenopodium rubrum. Physiol Plant 118:605–612
Letham DS (1971) Regulators of cell division in plant tissues.
A cytokinin bioassay using excised radish cotyledons.
Physiol Plant 25:391–396
Manchester LC, Tan DX, Reiter RJ, Park W, Monis K, Qi W
(2000) High levels of melatonin in the seeds of edible
123
Plant Growth Regul (2008) 55:29–34
plants. Possible function in germ tissue protection. Life
Sci 67:3023–3029
Murch S, Saxena PK (2002) Melatonin: a potential regulator of
plant growth and development? In vitro Cell Dev Biol
Plant 38:531–536
Murch S, KrishnaRaj S, Saxena PK (2000) Tryptophan is a
precursor for melatonin and serotonin biosynthesis in
in vitro regenerated St. John’s wort (Hypericum perforatum L. cv. Anthos) plants. Plant Cell Rep 19:698–704
Reiter RJ (1993) The melatonin rhythm: both a clock and a
calendar. Experientia 49:654–664
Reiter RJ, Tan DX, Manchester LC, Qi W (2001) Biochemical
reactivity of melatonin with reactive oxygen and nitrogen
species. Cell Biochem Biophys 34:237–256
Thomas JC, Katterman F (1992) 5-Bromodeoxyuridine inhibition of cytokinin induced radish cotyledon expansion.
Plant Sci 83:143–148
Van Tassel DL, Roberts NJ, Lewy A, O’Neill SD (2001)
Melatonin in plant organs. J Pineal Res 31:8–15
Wolf K, Kolar J, van Dongen W, van Onckelen H, Machackova I (2001) Daily profile of melatonin levels in
Chenopodium rubrum depends on photoperiod. J Plant
Physiol 158:1491–1493