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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 123 30 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) 123 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 123 32 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 123 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 33 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. 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