Comparisons of Scutellaria baicalensis, Scutellaria lateriflora and Scutellaria racemosa: Genome Size, Antioxidant Potential and Phytochemistry. Planta Med 74: ...
Melatonin in plants - scientific and practical aspects POSMYK Malgorzata M., JANAS Krystyna M. Department of Ecophysiology and Plant Development, University of Lodz, ul. Banacha 12/16, 90-237 Lodz, POLAND Abstract: A widespread occurrence of melatonin (Mel) in plant kingdom has been recently reported. Mel - till now known as an animal hormon and neurotransmiter - seems to be a highly conserved molecule occurring in evolutionary distant organisms. Its role in plants seems to be similar in some aspects to that in animals. Although Mel function in plants is not well known yet a hypothesis can be put forward that it probably acts as a ºnight signal, coordinating responses to diurnal and photoperiodic environmental cues. It has also been suggested that Mel is an independent plant growth regulator, probably its action is analogous to indole-3-acetic acid (IAA) and it may mediate the activities of other plant growth regulators. Due to its antioxidant properties Mel may also stabilise cell redox status and protect tissues against reactive oxygen species (ROS) and other harmful environmental influence. Some researchers speculate that Mel could also be used to improve the phytoremediation efficiency of plants against different pollutants.We have suggested that exogenous Mel applied into the seed could be a good biostimulator improving seed germination and seedling growth in abiotic stress conditions. Since Mel is safe to animals and humans as well as inexpensive, this technique could be a good, feasible and cost-effective tool for positive seed quality modification and may have economically important meaning for ecological agriculture. Keywords: abiotic stress, biostimulators, melatonin, priming.
1. Melatonin in Plants Melatonin (Mel) is a conserved substance, which was discovered in the evolutionary distant organisms: bacteria, mono- and multicellular algae, higher plants, invertebrates and vertebrates [1-4]. In vertebrates Mel is mainly produced by pineal gland and secreted into the blood stream in a rhythmic manner. Mel is an animal hormone involved in the regulation of physiological processes, including circadian rhythm and photoperiodis [5]. In animals and humans, Mel has been identified as a remarkable molecule signaling not only the time of day or year, but also promoting immunomodulatory and cytoprotective properties. In 1991 Science published a paper about the presence and role of Mel in photosynthesizing Gonyaulax polyedra and this inspired search for this substance in other authotrophic organisms [6] which led to its discovery in many higher plants [7-9]. This indoleamine was found in many organs of higher plants: roots, stems, leaves, flowers, fruits and seeds at concentrations usually ranging from picograms to nanograms per gram of tissue [8, 10-12]. Mel concentrations differ not only from species to species but also among varieties of the same species [7, 8, 13, 14]. Its highest level was found in the reproductive organs, most of all in flowers and seeds which may be related to their high sensitivity to environmental stresses e.g. UV irradiation [15]. The average Mel levels in seeds fall in the range 2-200 ng g -1 dry weight (DW) [3]. Manchester et al. [9] suggest that variations in Mel contents might result from different environmental impact during plant growth and development and during consecutive stages of seed morphological and physiological development. However, the high concentration of Mel detected in seeds presumably provides antioxidative defense in a dormant and more or less dry system, in which enzymes are poorly effective and cannot be upregulated. Thus in seeds Mel may be essential in protecting germ and reproductive tissues of plants from harmful environmental conditions. Mel was found in most of 108 medicinal herbs commonly used in traditional Chinese medicine. It was very interesting that the highest Mel concentrations were observed in the herbs used to retard aging and to treat diseases which associate with free radicals (e.g. neurological disorders) [16]. Mel contains an indole heterocycle and two side chains, namely a 5-methoxy group and 3-amide group. N-acetyl-5methoxytryptamine (Mel) is synthesized from the amino acid precursor, L-tryptophan (Fig. 1). A similar way of Mel biosynthesis in plants, mammals, yeast and bacteria seems to be supported by the presence of serotonin (5-hydroxytryptamine) which was found in fruit and vegetables more than 30 years ago. Also research on in vitro cultures of St. John’s wort (Hypericum perforatum L.) with the use of 14C-tryptophan revealed that serotonin synthesis occured via 5-hydroxytryptophan as in mammals [17, 18].
-2-
Fig. 1: Biosynthetic pathway of melatonin in plants [12]. Enzymes involved: Trp5H - tryptophan 5-hydroxylase (EC 1.14.16.4); AADC - aromatic L-amino acid decarboxylase (EC 4.1.1.28); TrpDC - L-tryptophan decarboxylase (EC 4.1.1.28); T5H - tryptamine 5-hydroxylase; TDA - tryptamine deaminase (EC 1.13.11.11); SNAcT - serotonin N-acetyltransferase (EC 2.3.1.87); AcSNMT acetyloserotonin N-metyltransferase (EC 2.3.1.4); HIOMT hydroxyindole-O-methyltransferase (EC 2.1.1.4). With the numbers basic tryptophan dependent patways of IAA biosynthesis with intermediates: [1] indole-3- pyruvic acid (IPA) and indole-3- acetaldehyd (IAAld), [2] indole-3-acetamid (IAM), and [3] indole-3acetonitrile (IAN) are marked.
In plants L-tryptophan is not only the precursor of serotonin and Mel but also of indole-3-acetic acid (IAA) and of other auxins e.g. indole-3-butric (IBA) and p-hydroxyphenylacetic acids [17,
19]
. IAA may be synthesized via several L-tryptophan dependent
pathways (Fig. 1) with [1] indole-3- pyruvic acid (IPA) and indole-3- acetaldehyd (IAAld), [2] indole-3-acetamid (IAM), or [3] indole-3- acetonitrile (IAN) as intermediate products [20]. However, Murch and Saxena [3] suggested that alternatively IAA could be synthesized also from tryptamine catalyzed by tryptamine deaminase (TDA) and this intermediate is directly connected with serotonin and Mel biosynthesis, too (Fig.1). The dominant Mel biosynthesis pathway seems to be that of tryptophan via 5-hydroxytryptophan to serotonin and than Nacetylserotonin formed by SNAcT and coverted to Mel by hydroxyindole-O-methyltransferase (HIOMT) with use S-adenosyl-Lmethionine (SAM) as a methyl group donor (Fig. 1) [3,
21]
. However, an alternative sequence of biosynthesis: serotonin via 5-
methoxytryptamine to Mel is also possible (Fig. 1, dashed arrow) [4]. In animals Mel is involved in the regulation of the circadian rhythm. It was called the hormone of darkness as its highest level is observed at night and during the day it decreases to the hardly detectable level. A similar pattern of Mel synthesis is observed in some Dinoflagellates and in some photosynthesizing plants [3]. However, very little is known concerning the physiological role played by Mel in plants [22, 23] although inconclusive attempts have been made to seek a role for this indolic compound as aºphotoperiodic and circadian regulator [14, 24, 25]. The amount of Mel is higher in young, reproductive tissues and falls down during senescence similarly as in animals. In young 2-day old seedlings of Pharbitis nil the content of Mel is 6 times higher then in older ones
[24]
. In etiolated Hypericum perforatum L.
[3]
Mel content is 15-20 times higher in comparison to those growing in the light . Thus, it seems that the high Mel content in the young seedlings growing in darkness may result from the combination of these two factors. Mel is believed to mediate the photoperiodic response in higher plants including the photoperiodic induction but the mechanisms are still unknown [3, 26]. The stimulatory role of Mel in plant growth was shown by Murch et al., [27] who used metabolism inhibitors of auxins, serotonin and Mel and observed that the increase in the endogenous Mel concentration induced root growth in Hypericum perforatum L . in vitro cultures while accumulation of serotonin – the direct Mel precursor – promoted shoot formation. It was reported that Mel stimulated vegetative growth of etiolated Lupinus albus L. hypocotyls in a manner similar to IAA
[28]
.
Mel and IAA were also seen to be distributed in lupine tissues in a similar concentration gradient. At high concentrations Mel acts as
-3an inhibitor (probably reaching the toxic level in tissues) while at lower concentration induces the growth of L. albus hypocotyl segments but not so effectively as IAA. Mel affects also the regeneration of lateral and adventitious roots in etiolated seedlings of L. albus, which was observed by comparing the effect of different concentrations of Mel and IAA on root promotion
[29]
. It was also confirmed that Mel acts as
a growth promoter in coleoptiles of wheat, barley, canary grass and oat, however its activity in comparison with IAA ranges between 10 and 55% [30]. Of particular note is similar inhibition effect of Mel and IAA on monocot root growth [30]. Mel has strong antioxidant properties [31,32]. Since Mel is soluble both in water and lipids it may be a hydrophilic and hydrophobic antioxidant. This fact together with Mel small size makes it particularly able to migrate easily between cell compartments in order to protect them against excessive ROS. In many studies Mel was observed to reduce oxidative damage of important molecules such as nucleic acids, proteins, lipids[33]. Its antioxidant activity seems to function via a number of means: (i) as a direct free radical scavenger, (ii) by stimulating antioxidant enzymes, (iii) by stimulating the synthesis of glutathione, (iv) by its ability to augment the activities of other antioxidants (v) by protection of antioxidant enzymes from oxidative damage (vi) by increasing the efficiency of mitochondrial electron transport chain thereby lowering electron leakage and thus reducing free radical generation [34-37]. Moreover, recent evidence indicates that the original Mel metabolite N1-acetyl-N2-formyl-5-methoxykynuramine (AFMK) which has been just identified for the first time in a plant Eichornia crassipes (water hyacinth) by Tan et al. [38] posseses high antioxidant abilities. It is now documented that the free radical scavenging capacity of Mel extends to its secondary, tertiary and quaternary metabolites [39]. It appears that Mel interaction with ROS is a prolonged process that involves its many derivatives. This process is referred to as the free radical scavenging cascade, which makes Mel highly effective, even at low concentrations, in protecting organisms from oxidative stress. Mel protects plant tissues against oxidative stress caused by harmful environmental factors such as: ozon concentration, high and low temperature, UV radiation, pollution [10, 40]. Mel was observed to be elevated in alpine and mediterranean plants exposed to strong UV irradiation, a finding amenable to the interpretation that Mel’s antioxidant properties can antagonize damage caused by light-induced oxidants
[15, 41]
environmental pollutants
. High levels of Mel exist in an aquatic plant, the water hyacinth, which is highly tolerant of
[38]
. Elevated levels of Mel probably help plants to protect themselves against environmental stress caused
by water and soil pollutants. Ta n et al.
[42]
also investigated the potential relationships between Mel supplementation and
environmental tolerance of plants. Their results showed that Mel added to the soil significantly enhanced pea plants tolerance to the copper contamination and increased their survival under previously lethal copper concentration. This is in agreement with the data obtained by Posmyk et al.
[43]
where pre-sowing seed treatment with Mel also protected red cabbage seedlings against toxic copper
ion concentrations. In ripe tomato fruit the level of Mel is much higher than that in green ones. It may be connected with protection of fruit against high ROS generation during ripening [7]. Also according to Van Tassel et al. [10] Mel may be involved in the maintenance of a proper organ redox state also during fruit ripening. Indoleamines are important for detoxification of reproductive organ tissues. It was observed that serotonin present in walnut seeds takes part in detoxification of excess ammonium which accumulates during dehydration and seed desiccation
[3]
. It seems that
because of lipophilic character and antioxidant properties Mel may protect lipids stored in seeds against peroxidation, thus increasing seeds viability and vigor [9, 10]. There are some reports about Mel being involved in seed dormancy [6] but the mechanisms of this regulation are unknown. However, ROS was defined as one of the factors regulating dormancy e.g. by protein oxidation and changes in protein carbonylation patterns [44] therefore, it is possible that Mel as the antioxidant regulating ROS level can indirectly influence seed dormancy. The role and biochemical mechanisms of Mel action in plant have not been clearly elucidated (Fig. 2). Does Mel act as a hormone independently or in concert with auxin and its own precursors and metabolites? The research on phyto-Mel and phytoserotonin is still in its infancy and little is understood of their roles in plant growth and development.
-4-
Fig. 2. The phyto-melatonin role and mechanism of action [12].
Recent plant neurobiology trends in science based on auxin signaling investigations [45] draw our attention to Mel as a molecule that has been known as a hormone and neurotransmitter in animal physiology and on the other hand, demonstrates parallelism with plant auxin - IAA. Resumption of the research on classic plant models used in plan physiology is necessary in order to try to clarify the role and mechanism of action of Mel (i) as an independent plant growth regulator, (ii) as a factor mediating the activity of other substances influencing plant growth or (iii) as a substance involved in growth regulation but whose activity generally is ascribed to other compounds. Firstly, Mel binding and interactions with Ca 2+-calmodulin have been shown to regulate many Ca2+-dependent cellular functions in animal cells thus Mel action in plants via Ca2+-calmodulin is a potential mechanism of signaling in plants particularly in view of the Ca2+-dependent activity of auxin in several physiological responses. Secondly, Mel may balance the oxidative stress generated by explanting and exposure to growth regulators or environmental factors during the culture process. Plants utilize ROS as second messengers in signal transduction cascades involved in diverse physiological functions including cell division, senescence, and tropisms [46]. Moreover, oxidative stress was earlier shown to stimulate cell division, differentiation, and morphogenesis analogous to auxins [47]. Mel and its metabolites in animal cells are known to trigger a cascade of free radical scavenging activities and a similar mode of action in plants is conceivable. On the basis of the above described hypotheses and Tan’s et al.[38, 42] preliminary data, we speculate that Mel could be used to improve the phytoremediation efficiency of plants against different pollutants. Since Mel is safe to animals and humans as well as inexpensive this may be a feasible and cost-effective approach to clean environmental contaminations. Further research in this area could provide valuable information on the significance of plant derived dietary supplements, agriculture and environment phytoremediation, however presented knowledge inclined to belive that melatonin caould be used as an effective biostymulators.
2. Biostymulators Plant crop production methods based only on improving agricultural technology (e.g. tillage, recultivation, fertilisation, irrygation, ect.) are limited due to the inability to effectively use the biological potential of the cultivated variety. Plant production and protection face a difficult task of preventing damage in field crops caused by harmful organisms or abiotic stresses - this way stimulating plant growth and devolopment, and reducing hazards presented to humans and environment as well as securing production of high quality and safe agricultural products at the same time. An urgent need arises of alternative ecological methods elaboration and of serch for new biologicaly active, ednvironmental friendly, safe substances, the best solution seems to be application of biostymulators .
-5Plant biostymulators – phytostymulators, are a different kind of non-toxic subsances of natural origin that improve and stimulate plant life processes otherwise than fertilisers or phytohormones. Their influence on plant is not the consequence of direct metabolic regulation properties but their action could be multidirect and they influence metabolism more generaly (Table 1). The crucial point is that biostymulators in contrast to bioregulators improve plant metabolic processes without changing their natural pathway. Tabele 1: Specyfication of biostimulators properties in contrast to bioregulators. Name Kind of substances Characteristic
• • •
•
BIOREGULATORS
BIOSTYMULATORS
Phytohormones
Phytostimulators
Auxins, Giberelins, Cytokinins, ABA, JA, Ethylene, Brassinosteroids
Different substances of natural origin (could be also syntezised), their mixtures, bio-extracts
Natural (usually secondary metabolites) or synthetic substances Not nutritional elements Regulate directly plant metabolism on molecular, cytological levels as well as in a whole plant - regulate plant growth and development Act at low concentrations
•
Are transprted from the place of their synthesis to the action sites in plant
•
Improue plant life processes while exogenous application of phytohormons can modify natural pathways of plant development (e.g. induction of fruit parthenogenesis, callus cultures or plant cell culture in vitro)
• •
Non-toxic, safe for human and environment substances Improve plant growth and development
•
Improuve plant life processes without modification of their natural pathways
•
Supply beneficial elements or organic compounds ready for use by plants
•
Indirect influence on metabolism e.g by different enzymes activity stimulation or inhibition, by influence on bioregulators synthesis.
•
Can act as antioxidants, osmoprotectants, elicitors
Modern, helth-promoting agriculture provokes an urgent need to elaborate of alternative safe ecological methods. A tendecy in plant protection is growing towards broader use of biological methods based on non-toxic plant origin substances, having different mode of action. They usually are not directly active against the harmful organisms as pesticides are, but they induce in plants certain immunity/resistance to patogens [48]. The results of Goëmar Labs. have shown that algae filtrates from Ascophyllum nodosum stimulate growth and nutrition of the treated plants and laminarin extracted from Laminaria digitata induces natural defense reactions [49]. The studies of their mode of action show that these products act as phytoactivators/biostymulators. Firstly, the filtrates stimulate the nitrate reductase and root phosphatases, involved in both nitrogen and phosphorus nutrition. Such stimulation results in better plant growth and increases chlorophyll content. Moreover, alga homogenate increases free polyamine content in plant tissues and it is especially important for better fruit set. Secondly, laminarin is a natural β-1,3-1,6 glucan extracted from brown alga and, as it is known, some of the fungal βglucans can be involved in the plant defence mechanisms – pay an important role as activators of natural plant resistance. Concidering the structural similarity between laminarin and fungal β-glucans the potential of laminarin to elicit/induce a cascade of natural defence responces causing plant resistance against phytopathogens was investigated and proved. Indeed, laminarin is clearly devoid of any direct antimicrobial activity but it induces resistance by plant defence activation [49]. Also betaines act as typical elicitors – factors which induce systemic acquired resistance to pathogens or other stresses in plants. The recommended biostymulators induce resistance in plants hence reduce the need of conventional treatments with synthetic chemicals. The others supply beneficial elements as e.g. titanium [50] or organic compounds ready for use by plants as e.g. aminoacids [51], poliphenols [52] economising energy for other needs as recovery processes after stress. Phenolic compounds influence lots of biochemical and physiological processes in plants [53]. Components of Asashi SL – comertialy available mixture of: sodium orthoand para- nitrophenolates with sodium 5-nitroguayacolate are easily metabolised by plants into other phenolics which are involved in mitochondrial enery-generating processes and their action results also in low cytoplasma viscosity which fosters translocation of all biosyntesis products. Moreover, diphenols are specific inhibitors of auxin oxidases so indirectly they have positive influence on this important phytohormone activity [54]. Being antioxidants as e.g. anthocyanins [55] or osmoprotectants as e.g. proline [56] biostimulators can provoke plant tolerance for unfarorable environmental conditions as chilling, drought, salinity, chemical contamination, heavy metal stresses. Nowadays, biostimulators are used in Poland in agriculture, floriculture, horticulture, on vegetable plantations and in orchards.
-6They are commertially available synthetic mixture of phenolic compounds e.g. Asahi SL or Atonik SL, but also natural ecologically preferable substances can be found: plant extract e.g. Adbios 850 SL – the mixture of oxyethyleneamines from coconut oil and methylesters from canola oil, alga extracts e.g. AlgaminoPlant – extract from brown alga supplemented with aminoacid composition; or complexes of humic and fulvic acids extracted from laminarite – HumiPlant. Many scientists as well as breeders consider biostimulator application as the most prospective and promssing method to produce ecological crops, to protect the enviroment, and to support safty-food production.
3. Biostymulator Application Methods Phytostimulators are usually used as supplementation with irigation (into foliage) or in substantia with fertilisers (into roots). They coud be also added to the medium in hydroponical cultivation [57]. The application moment is very important. As the technology mode this substances are implemented at crutial for coming yield quality and quantity stages of plant development. This way is very effective for fruit and vegetable production if relevant biostimulators are applied during flowering period and/or fruit generation. Biostimulators are also recommended as an intervention method and use in case of stress conditions e.g. black frost, drough, hail, strong wind, chemical contamination with herbicides or pesticides. They can be applied befor expected stress, during unfavorable condition and also after them [57]. Primary and basic condition determinating good and great harvest in spe is the quality of implemented seed material. The aim of our experiments was to find effective methods for biostymulator application into the seed to improve sowing material. The known technics of seed priming: hydro- and osmo- conditioning were tested. Different pre-sowing seed treatments effectively counteract diseases and pests as well as improve seed viability and seedling vigour per se [58]. They are based on understanding of the physiology, biochemistry and anatomy of plants that explain mechanisms of bioprocessess and give them practical application. The priming/conditioning methods are based on controlled seed hydration using (a) low water potential of active osmotic solutions (osmopriming) or matripotential of carriers (matriconditioning); (b) high air humidity (drumpriming); (c) water soaking (hydropriming) leading to seed imbibition to prepare them for sowing in a gel (fluid drilling) [58, 59]. This techniques can be combined with other supporting factors such as aeration, light-irradiation, temperature-stratification. Seed priming can also be combined with application of growth regulators and other bioactive substances [59]. Generally, priming is accompanied by an increase in the activities of numerous enzymes, e.g., phosphatases, synthases, peroxidases and other antioxidant enzymes [60]. Intensification of protein and nucleic acid metabolism is also observed Bray et al [61]. Moreover, priming facilitates cell membrane structure reorganisation from hexagonal (in dry seeds) to lamellar (in imbibed seeds). This reorganization is necessary for biophysical and biochemical processes during germination and accelerates seedling emergence[62]. Priming allows plants to survive environmental stresses since it improves their recovery potential.
4. Seed Priming with Melatonin – Advantages in Abiotic Stress Conditions Recent plant neurobiology trends in science based on auxin signaling investigations
[45]
focus our attention on melatonin –
a molecule that has been known as a hormone and neurotransmitter in animal physiology, and which demonstrates parallelism with plant auxin, IAA. Resumption of the research on classic plant models used in plant physiology is necessary to clarify the role and mechanism of action of melatonin: (i) as an independent plant growth regulator, (ii) as a factor mediating the activity of other substances influencing plant growth or (iii) as a substance involved in growth regulation but whose activity generally is ascribed to other compounds whereas our recent knowledge qualifies Mel as a biostimulator. Generally, as it was mentioned before, the physiological concentrations of Mel in the seeds studied were very high, for example in white and black mustard seeds it was 129 and 189 ng g-1, respectively [8]. This level of Mel is much higher than the known physiological concentrations in the blood of many vertebrates. Since the seed, particularly its germ tissue, is highly vulnerable to oxidative stress and damage, we surmise that the high concentrations of Mel detected in seeds presumably provide antioxidative defense. Indeed, exogenous Mel application by hydropriming into the red cabbage seeds was a good tool for seed vigor improvement [55]. Positive effect of this treatment was visible especially under copper stress conditions. Similarly, experiments with cucumber seeds osmoprimed with Mel proved the thesis about its positive effects on seeds [63]. The seeds osmoprimed with Mel started to germinate at 1 0 oC – which was impossible for the control, untreated ones. Increasing scientific evidence apeares that Mel in seeds may be essential in protecting germ and reproductive tissues of plants from oxidative damage caused by ultraviolet light, drought, extremes
-7in temperature, and environmental chemical pollutants
[9, 15]
. Currently, on ISEST 2009, two posters concerning Mel application into
corn seeds by hydropriming and into cucumber seeds by osmopriming as well as its influence on subsequent seedling growth in abiotic stress conditions are presented. These were the laboratory experiments, we are incleaned to belive that fild tests are absolutely necessary.
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