Melatonin in Plants and Plant Culture Systems: Variability, Stability and Efficient Quantification

Abstract

Despite growing evidence of the importance of melatonin and serotonin in the plant life, there is still much debate over the stability of melatonin, with extraction and analysis methods varying greatly from lab to lab with respect to time, temperature, light levels, extraction solvents, and mechanical disruption. The variability in methodology has created conflicting results that confound the comparison of studies to determine the role of melatonin in plant physiology. We here describe a fully validated method for the quantification of melatonin, serotonin and their biosynthetic precursors: tryptophan, tryptamine and N-acetylserotonin by liquid chromatography single quadrupole mass spectrometry (LC-MS) in diverse plant species and tissues. This method can be performed on a simple and inexpensive platform, and is both rapid and simple to implement. The method has excellent reproducibility and acceptable sensitivity with percent relative standard deviation (%RSD) in all matrices between 1 and 10% and recovery values of 82–113% for all analytes. Instrument detection limits were 24.4 ng/mL, 6.10 ng/mL, 1.52 ng/mL, 6.10 ng/mL, and 95.3 pg/mL, for serotonin, tryptophan, tryptamine, N-acetylserotonin and melatonin respectively. Method detection limits were 1.62 μg/g, 0.407 μg/g, 0.101 μg/g, 0.407 μg/g, and 6.17 ng/g respectively. The optimized method was then utilized to examine the issue of variable stability of melatonin in plant tissue culture systems. Media composition (Murashige and Skoog, Driver and Kuniyuki walnut or Lloyd and McCown's woody plant medium) and light (16 h photoperiod or dark) were found to have no effect on melatonin or serotonin content. A Youden trial suggested temperature as a major factor leading to degradation of melatonin. Both melatonin and serotonin appeared to be stable across the first 10 days in media, melatonin losses reached a mean minimum degradation at 28 days of approximately 90%; serotonin reached a mean minimum value of approximately 60% at 28 days. These results suggest that melatonin and serotonin show considerable stability in plant systems and these indoleamines and related compounds can be used for investigations that span over 3 weeks.

Melatonin‐deficient rice plants show a common semidwarf phenotype either dependent or independent of brassinosteroid biosynthesis

“…SNAT is highly conserved in plant species ranging from cyanobacteria to higher plants, indicative of the essential role of SNAT in melatonin synthesis and offering indirect evidence of the omnipresence of melatonin in the plant kingdom. The few plants tested so far containing undetectable levels of melatonin possess SNAT genes in their genomes, suggesting that these plants also have the capacity to synthesize low levels of melatonin …”

Analysis and measurement of serotonin

“…High‐performance liquid chromatography coupled to mass spectrometry (LC/MS) has become a widely used and sensitive technique for the analysis of serotonin and its metabolites. For example, single quadrupole LC/MS was utilized for the analysis of serotonin and melatonin in plants (Erland, Chattopadhyay, Jones, & Saxena, ). Atmospheric pressure ionization techniques such as electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI) have been used in LC/MS to analyze serotonin and its metabolites in a variety of samples (Carrera, Sabater, Vilanova, & Sogorb, ; Park et al, ).…”

“…In another study, LC/MS with APCI was employed for the analysis of serotonin and catecholamines in bovine chromaffin cells and achieved comparable sensitivity with no derivatization (Carrera et al, ). LC/MS systems are relatively inexpensive to use for routine analyses (Erland et al, ), but their ability to provide structural information and selective detection is limited compared with the more sophisticated triple quadrupole LC/MS systems (Kuepper, Musshoff, & Madea, ; Schreiber, ).…”

“…A validated LC/MS method was implemented for a detailed analysis of serotonin and melatonin in several plant samples such as the roots, shoots and seeds of St John's wort, banana, potato, tobacco, elm, fennel and other plants (Erland et al, ). Moreover, circadian and seasonal changes in serotonin and melatonin levels were reported in Chara australis Brown, a member of the Characeae family, after analysis with a LC/MS/MS method (Beilby, Turi, Baker, Tymm, & Murch, ).…”

“…GC/MS methods employing single or triple quadrupole MS technology with electron impact ionization were used to measure serotonin and its metabolites in standard solutions (Suñol & Gelpi, 1977) and human urine (Naccarato et al, 2014) and a GC/MS method with negative ion chemical ionization was used for the analysis of serotonin in human and rat plasma (Sano et al, 2014). PFPA in ACN~1 ng Suñol and Gelpi (1977) (Erland, Chattopadhyay, Jones, & Saxena, 2016).…”