Growth conditions determine different melatonin levels in Lupinusalbus L.

Abstract: Melatonin, an indoleamine, which has recently been assigned several

roles in plant physiology as a growth promoter, as rooting agent, and as

antioxidant in senescence delay and cytoprotection, seems to have a relevant

function in plant stress situations. The presence of melatonin increases the

resistance of lupin plant tissues (Lupinus albus L.) against natural or artificially

induced adverse situations. In this work, we studied the response of lupin

plants in controlled stress situations (drought-, anaerobic-, pH-, and cold

stress and using ZnSO4, NaCl, and H2O2 as chemical stressors) and measured

the changes in endogenous melatonin levels in lupin plants. Also, the effect of

abscisic acid, ethylene, and natural environmental conditions were evaluated.

In general, nearly all stressful factors caused an increase in melatonin in the

investigated organs. The chemical stress provoked by ZnSO4 or NaCl caused

the most pronounced changes in the endogenous level of melatonin, followed

by cold and drought stressors. In some cases, the level of melatonin increased

12-fold with respect to the levels in control plants, indicating that melatonin

biosynthesis is upregulated in common stress situations, in which it may serve

as a signal molecule and/or as a direct antistress agent due to its well-known

antioxidative properties.

Marino B. Arnao and JosefaHern�andez-Ruiz

Department of Plant Physiology, Faculty of

Biology, University of Murcia, Murcia, Spain

Key words: abiotic stress, antioxidant, chemical

stress, lupin, Lupinus albus L., melatonin, plant

stress

Address reprint requests to Marino B. Arnao,

Department of Plant Physiology, Faculty of

Biology, Campus of Espinardo, University of

Murcia, 30100-Murcia, Spain.

E-mail: marino@um.es

Received February 15, 2013;

Accepted March 22, 2013.

Introduction

Melatonin (N-acetyl-5-methoxytryptamine) was first iden-

tified in vascular plants in 1995 by two research groupssimultaneously [1, 2]. Previously, the first reference to thepresence of melatonin in a photosynthesizing organism

was made by Poeggeler et al. [3], in the unicellular algaLingulodinium polyedrum Stein. Since then, this indole-amine has been detected in a wide range of plant species,

including cereals, vegetables, fruits, and roots. Indeed,plant tissues seem to contain much higher melatonin levelsthan are observed in animals. In plants, the levels vary

substantially, from picograms to micrograms per gram offresh weight (FW) [4–9]. However, there have been fewcontrols on the physiological state of plant tissues understudy and its influence on endogenous melatonin levels.

It has been proposed that melatonin plays several rolesin plants [1, 4, 7, 10–14]. For example, some studies havedemonstrated the action of melatonin as growth promoter,

in a similar way to the plant hormone indole-3-acetic acid(IAA) [15–18]. It acts as rooting agent in different speciessuch as lupin, cucumber, cherry, and rice [19–23], where it

shows some similarities with IAA, but also has importantpeculiarities in its action [24]. Many works have focusedon the role of melatonin as cytoprotector and abioticstress protector because of its relevant properties as a scav-

enger of free radicals, especially reactive oxygen and nitro-gen species [25, 26]. In this regard, the apoptoticprevention afforded by melatonin in cultured carrot cells,

the delay in senescence brought about by melatonin inbarley, rice and, apple leaves, and its evident protective

role against chemical stresses should be cited as examplesof some significant cases.A possible response of plants in stress situations may be

to increase their endogenous melatonin content. However,the influence of environmental factors such as sunlight,wind, fluctuating temperatures, soil type, air humidity, air

contaminants, and pathogens on the melatonin content ofplant tissues has not generally been taken into account.Only recently, have some studies considered this subject,

which would appear to be an important issue from anagricultural and nutritional perspective [14, 27].In this article, we studied the influence of various

physico-chemical agents on the levels of melatonin inwhite lupin (Lupinus albus L.) plants. The chemical stres-sors such zinc, sodium chloride, and hydrogen peroxidewere also used; also, plants were exposed to drought-,

anaerobic-, pH- and cold stress conditions. The effect ofphytoregulators [abscisic acid (ABA) and ethylene] isstudied with respect to the changes seen in the melatonin

content compared with control plants. The influence ofenvironmental factors on melatonin levels of plants grow-ing under artificial conditions is also presented.

Materials and methods

Reagents

Melatonin (N-acetyl-5-methoxytryptamine) was purchasedfrom Acros Organics Co. (Geel, Belgium). The solvents,

chloroform, water, and acetonitrile (HPLC grade), wereobtained from Scharlau Chemie (Barcelona, Spain). The

149

J. Pineal Res. 2013; 55:149–155Doi:10.1111/jpi.12055

© 2013 John Wiley & Sons A/S.

Published by John Wiley & Sons Ltd

Journal of Pineal Research

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reagents and salts (analytical grade) used were obtainedfrom Merck (Darmstadt, Germany).

Plant material

Lupin (Lupinus albus L.) seeds were sterilized in 10%hypochlorous acid solution for 5 min, washed three times

with distilled water, and then soaked in distilled water for24 hr at 24°C in darkness.

Standard growth conditions of lupin plants

Lupin plants were grown in pots (60 9 50 mm ø) with

vermiculite (an inert substrate) in a controlled chamber at24°C and 50–65% relative humidity for 9 days. The plantswere exposed to a standard photoperiodic cycle (16-hrlight/8-hr dark) with a photosynthetic photon flux density

(PPFD) of 80–90 lmol photon/m2/s supplied by 36W Syl-vania-Grolux fluorescent tubes. The PPFD was measuredwith a Delta OHM-HD9021 quantum sensor (Padua,

Italy).

Chemical stress

Intact 9-day-old plants in pots were treated through theroots, for 24 hr, at 24°C and in the light, with differentstress agents dissolved in 10 mM sodium phosphate-buf-

fered medium (pH 6.5): 10 mM hydrogen peroxide (H2O2),10 mM sodium chloride (NaCl), and 1 mM zinc sulfate(ZnSO4). Control plants were maintained only in buffered

medium. After 24 hr, roots were collected for melatoninestimation.

Drought stress

Intact 9-day-old plants in pots were treated in three differ-

ent irrigation modes: group A (three irrigations to fieldcapacity, 50 mL 9 3 of water); group B (two irrigations,50 mL 9 2 of water); and group C (only one irrigation,50 mL of water). After 7 days, roots were collected for

melatonin estimation.

Anaerobic stress

Intact 9-day-old plants were transferred to tubes contain-ing different levels of molecular oxygen in water: 0%,

20%, 50%, and 100%. The different percentages wereobtained through the mixture with solutions treated withpure N2 and O2. After 3 days, roots were collected formelatonin estimation.

pH adjustment

Intact 9-day-old plants were transferred to tubes contain-ing different buffered media, from pH 3.5 to 8.5. After3 days, roots were collected for melatonin estimation.

Cold stress

Intact 9-day-old plants in pots were maintained in agrowth chamber at 6°C in the standard photoperiod

conditions. Control plants were maintained at 24°C andthe same photoperiod. After different intervals (2, 18,24, and 36 hr), roots were collected for melatoninestimation.

Ethylene and abscisic acid effect

Intact 9-day-old plants were transferred to tubes and trea-ted through the roots, for 3 days, with the phytoregulatorsEthrelTM (2-chlorethylphosphonic acid; an ethylene genera-

tor) at 0.5 and 1.5 mM and ABA at 100 and 200 lM. Con-trol plants were maintained only in buffered medium.After 3 days, roots were collected for melatonin

estimation.

Exogenous melatonin

Intact 9-day-old plants were transferred to tubes and trea-ted through the roots with exogenous melatonin at con-centrations of 1, 5, and 10 lM. After 3 days, roots were

collected for melatonin estimation.

Controlled versus natural conditions

Intact 9-day-old plants were cultivated in two differentconditions: (a) in pots containing conventional substrate,for 30 days in a controlled growth chamber at 24°C,with 50–60% relative humidity and the standard photo-periodic cycle described above, and (b) 10 days as in (a)and 20 days in open field conditions, in installations of

the Agricultural Experimental Service of the Universityof Murcia, during July, 2011; during this month, the airtemperatures varied between 17°C and 39°C, with a

maximum PPFD of 2400 lmol photon/m2/s and a rela-tive humidity of 30–75%, according to the data collectedfrom the weather station at the same plot. After

30 days, roots and leaves were collected for melatoninestimation.

Melatonin measurements

To measure the melatonin content in plant samples adirect sample extraction procedure was used [9]. For

this, 0.5 g FW of tissue was cut into sections (3–5 mm)and, without homogenization, placed in vials containing3 mL of chloroform, overnight (15 hr) at 4°C in dark-

ness with shaking. The sections were discarded afterbeing washed with 0.5 mL of solvent and then evapo-rated to dryness under vacuum using a SpeedVac Ther-moSavant mod. SPD111V coupled to a refrigerated

RVT400 vapor trap (Holbrook, NY, USA). The dry res-idue was redissolved in 0.5 mL of acetonitrile, filtered(0.2 lm), and analyzed by LC with fluorescence detec-

tion. The procedures were carried out in dim artificiallight.These same procedures were applied to standard mela-

tonin solutions at different concentrations (0.01 and0.1 lM) to calculate the recovery percentage with the sam-ple extraction procedure. A recovery rate of 95% in spiked

samples was obtained and used for the final calculation ofmelatonin in plant tissues [8, 9].

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Arnao and Hern�andez-Ruiz

Melatonin analysis by liquid chromatography withfluorescence detection

A Jasco liquid chromatograph (Tokyo, Japan), a Waters

Spherisorb-S5 ODS2 column (250 9 4.6 mm), and a JascoFP-2020-Plus fluorescence detector were used to measuremelatonin levels. An excitation wavelength of 280 nm and

an emission wavelength of 350 nm were used. The iso-cratic mobile phase consisted of water/acetonitrile (60:40)at a flow rate of 0.2 mL/min. The data were analyzed

using the Jasco ChromPass 1.8.6 Data System Software.An in-line fluorescence spectral analysis (using the JascoSpectra Manager Software) compared the excitation and

emission spectra of standard melatonin with the corre-sponding peak in the samples.

Liquid chromatography with time-of-flight/massspectrometry

Identification of melatonin in plant extracts was confirmed

using an Agilent 6220 Accurate Mass TOF LC/MS (SantaClara, CA, USA) equipped with an electrospray interfaceoperating in positive ion mode. The LC system was an

Agilent 1200 series consisting of two pumps and a ZorbaxRapid Resolution C18 (2.1 mm 9 50 mm, d.f. 1.8 lm)column at a flow rate of 0.4 mL/min. A linear gradient of5–95% of acetonitrile in water with 0.1% formic acid for

15 min was used. The injection volume was 40 lL. Thefollowing operation parameters were used for theLC-TOF/MS measurements: capillary voltage, 2200 V; gas

temperature, 350°C; nitrogen drying gas set at 11 L/min;and nebulizer pressure of 45 psi. The dielectric capillaryexit was 120 V (fragmentor setting) and the skimmer

60 V. The octupole DC1 was set to 35.5 V and octapoleRF to 250 V. The instrument was tuned and calibratedwith the standard calibration solution provided by the

manufacturer with reference m/z of 149.02332 and322.048121. LC-TOF/MS accurate mass spectra wererecorded in the range 140–350 m/z at a scan rate of 1-s perspectrum. The data recorded were processed with the

Agilent MSD-TOF Software.

Statistical analysis

For the melatonin data, differences were determined usingthe SPSS 10 program (SPSS Inc., Chicago, IL, USA),

applying the LSD multiple range test to establishsignificant differences at P < 0.05.

Results

In previous studies, we confirmed the presence of melato-nin in several plants including lupin, oat, barley, wheat,

and canary grass, using LC-MS/MS with electrosprayionization in multiple reaction monitoring mode andLC-TOF/MS for identification [8, 15, 16]. Simultaneously,

melatonin was quantified using a fluorescence detectorcoupled to the LC, providing an in-line spectral analysiswith the respective excitation and emission spectra [9].

Figure 1 shows the effect of the different chemicalagents assayed on the endogenous melatonin level in lupin

plants. The three agents (H2O2, NaCl, and ZnSO4) pro-voked an increase in endogenous melatonin levels with

respect to control plants, the increase being, in all cases,dependent on the incubation time and the concentration(data not shown). As can be seen, ZnSO4 had the highest

stimulatory effect on the melatonin content, followed byNaCl and H2O2. Note that H2O2 and NaCl were used atconcentrations ten times higher than that of ZnSO4

(Fig. 1A). Fig. 1B shows how many times the endogenousmelatonin content increased over control levels for eachchemical stressor agent assayed (multiplication factor).ZnSO4 provoked a considerable increase in the melatonin

content of lupin roots, to around 80 ng/g FW (Fig. 1A),which was 12 times the melatonin content of the controlplants, while NaCl and H2O2 had a less pronounced effect.

A simple drought stress on lupin roots was generated bylimiting the irrigation water volume for 7 days. Table 1shows the melatonin content of plants irrigated in different

ways. The data clearly show that melatonin increased inthe roots of plants exposed to a water restriction. Thus,lupin plants irrigated only with 50 mL presented a consid-

erable accumulation of melatonin (56.8 ng/g FW)compared with control plants (13.9 ng/g FW; Table 1).Another stress situation was assayed by subjecting lupin

plants to diverse levels of oxygen availability. For this,

intact 9-day-old plants were transferred to tubes contain-ing different molecular oxygen levels in water. Four levelsof dissolved O2 were obtained: 0%, 20%, 50%, and 100%

of O2. Fig. 2 shows the melatonin content of roots at theseoxygen levels. As can be seen, O2 restriction in the assayedperiod (3 days) provoked an increase in the melatonin

content of roots.Lupin plants growing in buffered medium at pH 6.5 pre-

sented a melatonin content of around 7 ng/g FW (Fig. 3).Modification of the pH in the culture media provoked

slight modifications in the melatonin content of roots:alkalinization produced a decrease in the melatonincontent, whereas acidification resulted in an increased of

melatonin content (Fig. 3).

(A)(B)

Fig. 1. (A) Melatonin content of lupin roots after 24 hr of differ-ent treatments: control (buffer solution), 10 mM H2O2, 10 mM

NaCl, and 1 mM ZnSO4. (B) Multiplier effect, with respect tocontrol, that every treatment had on the melatonin content ofroots. Error bars represent standard errors of the mean (n = 12).Different superscripts indicate statistically significant differences atP < 0.05.

151

Stress and melatonin content of lupin

Another relevant factor in plant development is the

ambient temperature. Fig. 4 shows the changes measuredin the melatonin content of roots at two different tempera-tures. The melatonin content of lupin plants growing at

6°C was significantly higher than that of plants growing at24°C. This melatonin increase was also time dependent,reaching a maximum after 36 hr (Fig. 4).Intact 9-day-old plants were treated through the roots,

for 3 days, with EthrelTM(2-chlorethylphosphonic acid; anethylene generator) at 0.5 and 1.5 mM and with ABA at100 and 200 lM. Table 1 shows the effect of these phyto-

regulators versus control plants with respect to melatonincontent. All the treatments led to an increase in the mela-tonin content of the roots, the level doubling when plants

were treated with 0.5 mM EthrelTM.

The melatonin content in roots was also be increased byincubating intact plants with exogenous melatonin, as can

be seen in Table 1, which shows the effect of melatonintreatment (1, 5 and 10 lM) on the melatonin content ofroots.Lastly, we measured the melatonin content of plants

cultivated for 30 days in two different conditions: in a con-trolled growth chamber at 24°C, with 50–60% relativehumidity and the standard photoperiodic cycle described

above, and in open field conditions as described in theMaterials and Methods section. Table 1 shows that theroots of plants growing in the open field had a melatonin

content three times the levels of plants cultivated in con-trolled conditions (growth chamber). In this case, we alsomeasured the melatonin content of leaves, which was seen

to be considerably higher (2.5 times) in the plants grownin the field.

Table 1. Melatonin content of lupin plants in different conditions

Melatonin content of roots(ng/g FW; �SE, n = 5)

Water stressThree irrigation (150 mL) 13.9 � 0.8Two irrigation (100 mL) 39.4 � 2.6One irrigation (50 mL) 56.8 � 3.3ABA & ethylene effectControl 7.2 � 0.4ABA 100 lM 9.5 � 0.6ABA 200 lM 10.4 � 0.6Ethrel 0.5 mM 14.1 � 0.9Ethrel 1.5 mM 8.9 � 0.5Exogenous melatonin effectControl 7.7 � 0.5Melatonin 1 lM 37.3 � 2.5Melatonin 5 lM 58.9 � 3.4Melatonin 10 lM 180.0 � 12.3

Natural or controlled conditions Roots Leaves

In controlled chamber 18.3 � 1.6 30.2 � 2.0In open field 55.6 � 3.6 75.6 � 5.3

ABA, abscisic acid; FW, fresh weight.

Fig. 2. Melatonin content of lupin roots after 3 days of incubat-ing plants at different molecular oxygen levels in water. Error barsrepresent standard errors of the mean (n = 12). Different super-scripts indicate statistically significant differences at P < 0.05.

Fig. 3. Melatonin content of lupin roots after 3 days of incubat-ing plants in different buffered pH media. Error bars representstandard errors of the mean (n = 12). Different superscripts indi-cate statistically significant differences at P < 0.05.

Fig. 4. Melatonin content of lupin roots after different lengths (2,8, 14, and 36 hr) of temperature treatment (24°C and 6°C). Errorbars represent standard errors of the mean (n = 12). Differentsuperscripts indicate statistically significant differences atP < 0.05.

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Arnao and Hern�andez-Ruiz

Discussion

Of all the stressors evaluated, ZnSO4 provoked the great-est increase in the endogenous melatonin content of lupin

roots (Fig. 1). It is known that heavy metals, such as Cu,Zn, Ni, and Co, provoke a strong antistress response dueto the high toxicity of these elements in plants. The mela-

tonin biosynthesis induction has also observed when rootsections of barley (Hordeum vulgare L.) were treated withZnSO4, and the roots of barley plants treated for 24 hrwith 1 mM ZnSO4 show a sixfold increase in the melatonin

content [28]. The other chemical stressors evaluated (H2O2

and NaCl) also increased melatonin levels in lupin andbarley roots but to a lesser degree (Fig. 1 and [28]). These

responses in both lupin and barley were clearly dose- andtime dependent. However, the response was more rapid inlupin roots because barley roots needed 3 days (48 hr

more than lupin to show a similar response), suggestingthat lupin roots are a more sensitive material for studyingthe biosynthetic response of melatonin [28].

Several articles report the cellular protective role ofexogenous melatonin. For example, the reduction in cop-per toxicity by melatonin (1–10 lM) has been described inred cabbage (Brassica oleracea rubrum) [29]. Also, Tan

et al. [30] demonstrated that melatonin added to soilenhanced the tolerance and survival of pea plants (Pisumsativum L.) against copper contamination, indicating that

the presence of melatonin in plants can be used for phyto-remediation purposes. The three different chemicalsassayed in the present work provoked stress for different

reasons: ZnSO4 through toxic stress; H2O2 through oxida-tive stress; and NaCl through osmotic stress. However, allthree produced a similar response in the form of an

increase in melatonin levels.Another means by which they cause osmotic stress is to

lower the water potential through a deficiency of watersupplied to the roots. Lupin plants with a restricted water

supply increased their melatonin content in their roots sev-eral folds (Table 1), demonstrating that melatonin is alsobiosynthesized in drought situations. Consistent with this,

water-stressed cucumber plants (Cucumis sativus L.) trea-ted with exogenous melatonin (50–500 lM) showed a clearimprovement in parameters such as seed germination rate

[31], lateral root generation, root and shoot growth,photosynthetic rate, chlorophyll content, and stomatalconductance, indicating that the application of melatoninminimized the induced water stress [20]. For apple trees

(Malus domestica Borkh. var. Hanfu) subjected to long-term drought stress, exogenous melatonin treatmentthrough the roots provoked a marked reduction in leaf

senescence, improved photosynthetic parameters such asphotosystem II efficiency and enhanced the activity ofantioxidative enzymes, leading the authors to propose the

use of melatonin for agricultural purposes [32, 33].Restricting the oxygen supply to roots also provoked a

slight increase in root melatonin levels, compared with

oxygen-rich solutions after 3 days of treatment (Fig. 2).Also, alkalinization of the medium lowered the root mela-tonin content, whereas acidification resulted in a slightincrease of the same (Fig. 3). These data indicate that

roots are sensitive to changes in their rhizosphere environ-ment (pH, salinity, oxygen availability, toxic elements, andwater potential) suggesting that roots are a potential mela-tonin biosynthetic organ. This is supported by the exis-

tence of high melatonin levels in lupin roots and by theexistence of an evident concentration gradient of melato-nin, between apical and basal zones or between main (pri-

mary) and lateral (secondary) roots. Generally, highestmelatonin concentration is found in the most activelygrowing zones [8, 19].

Ambient temperature is a crucial factor in plant devel-opment. The response of plants to low temperature hasbeen well studied in the form of cold stress [34]. Lupin

plants in a cold environment (6°C) showed a 2.5-foldincrease in melatonin levels, compared with control plantsgrowing at 24°C (Fig. 4). This is another stress situationthat led to an increase in melatonin levels as a response.

The protective effect of exogenous melatonin during chill-ing stress has been described. For example, cucumberseeds treated with melatonin (25–100 lM) improved their

germination rate [31], and the presence of melatonin (40–80 nM) in Daucus carota L. cell suspension attenuatedcold-induced apoptosis [35]. Lastly, melatonin improved

the survival of cryopreserved callus of Rhodiola crenulata[36].Some plant hormones appear to be decisive in stress

responses. In this respect, the role of ethylene and ABA as

stress response mediators has been well studied. ABA isproduced by roots during drought stress. In flooded soilsituations, ethylene is generated in the root tissues through

the excessive accumulation of 1-aminocyclopropane-1-car-boxylic acid (the natural precursor of ethylene) because itstransportation to the stem is blocked. The treatment of

lupin plants with an artificial precursor of ethylene (Eth-relTM) or with ABA provokes, in both cases, an increase inthe melatonin level of roots (Table 1). These preliminary

data suggest that these plant hormones play a role in thebiosynthetic upregulation of melatonin caused by plantstress. In this respect, Pelagio-Flores et al. [24] recentlydemonstrated that melatonin can regulate the root

architecture of Arabidopsis thaliana independent of auxinsignaling.The possible influence of environmental conditions on

melatonin levels was studied in an experimental approachthat was intended to provide data on a crucial aspectfrequently overlooked and which may influence the mela-

tonin content of plants grown in laboratory conditions(indoors) compared with plants cultivated in field condi-tions. Field-grown plants are exposed to physical, chemi-cal, and biological agents, in natural abiotic and biotic

stress situations unlike plants grown in artificial conditionsin which light/dark, temperature, humidity, etc. are rigidlycontrolled in plant growth chambers. Plants grown

indoors, in moderate conditions, have a lower melatonincontent than those cultivated in the field with its more var-iable conditions (Table 1). Plants cultivated outdoors

showed three times more melatonin in roots and 2.5 timesin leaves than chamber-grown plants. These datashow that environmental conditions decisively affect the

melatonin content of tissues.

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Stress and melatonin content of lupin

Recently, we demonstrated this effect in tomato plants(Solanum lycopersicum L. var. Cherry) [27]. Leaves oftomato plants cultivated in outdoor conditions had tentimes more melatonin than leaves cultivated in vitro and

seven times more than leaves of plants cultivated in potsin a growth chamber. The tomato stems and roots showedthis same environmental influence. These data and others

point to the relevance of environment conditions on themelatonin content of plant tissues, an effect that may berelated to the antioxidative protection that melatonin

affords against harmful environmental agents [14]. Amongthese agents, solar radiation and UV radiation havereceived special attention. Dubbels et al. [1] were the first

to propose a role for melatonin as an antioxidant compar-ing the different susceptibility to ozone of two cultivars ofNicotiana tabacum L. Surprisingly, Glycyrrhiza uralensisroots increased their melatonin content to about 80 lg/gFW after UV-B treatment [37]. In Ulva sp., the melatonincontent increased when the macroalga was exposed to hos-tile weather or toxic metals, thus preserving chlorophylls

levels [38]. This chlorophyll protective effect of melatoninwas first described in barley leaves (Hordeum vulgare L.)in which exogenous melatonin application preserved the

chlorophyll content during dark-senescence induction [39].The water hyacinth Eichhornia crassipes also increasedmelatonin levels when grown in sunlight (outdoor condi-tions) compared with plants growing in growth chambers

[40].Some authors have pointed to differences in melatonin

levels between Alpine and Mediterranean plants, which

was attributed to the strong influence of high UV radia-tion [41]. In these cases, UV radiation appears to triggerthe increases in melatonin levels. However, the data

presented in this work show that other environmentalagents such as cold, drought stress, and temperature fluc-tuations, may also have a significant effect on the levels of

melatonin in plants.To conclude, the changes in endogenous melatonin lev-

els in lupin in response to the different controlled stress sit-uations studied show that practically all stress-producing

actions provoke an increase in the root melatonin content,indicating that biosynthesis of melatonin is clearly upregu-lated in common stress situations. Melatonin seems to act

as a signal molecule and as a direct antistress agent due toits antioxidative properties. However, although manyadvances have been made in our understanding of the mel-

atonin biosynthetic pathway in plants [42, 43], much aboutthe enzymes and their regulation remains unknown.

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Stress and melatonin content of lupin