Metabolic analysis of the melatonin biosynthesis pathway using chemical labeling coupled with liquid chromatography‐mass spectrometry

Abstract

Characterization of the melatonin (MLT) biosynthesis pathway in plants is still limited. Additionally, a metabolomic analysis of MLT biosynthesis in plants is still a challenge due to analyte structural and chemical diversity, low analyte abundances, and plant matrix complexities. Herein, a sensitive liquid chromatography‐mass spectrometry (LC‐MS) method enabling the simultaneous determination of seven plant MLT biosynthetic metabolites was developed. In the proposed strategy, the targeted metabolites, which included tryptophan (Trp), tryptamine (TAM), 5‐hydroxytryptophan (5HTP), serotonin (5HT), N‐acetylserotonin (NAS), 5‐methoxytryptamine (5MT), and MLT, were purified from plant extracts using a one‐step dispersive solid‐phase extraction (DSPE). The samples were then chemically labeled with dansyl chloride (DNS‐Cl), followed by analysis using LC‐MS. The limit of detection (LOD) values ranged from 0.03 to 1.36 pg/mL and presented a 22‐ to 469‐fold decrease when compared to the unlabeled metabolites. Due to the high sensitivity of the proposed method, the consumption of plant materials was reduced to 10 mg FW. Ultimately, the established method was utilized to examine the distributions of MLT and its intermediates in rice shoots and roots with or without cadmium (Cd) stress. The results suggested that under normal condition, MLT may also be generated via a Trp/TAM/5HT/5MT/MLT path (Pathway II) in addition to the previously reported Trp/TAM/5HT/NAS/MLT path (Pathway I), although Pathway I was shown to be dominant. During Cd stress, MLT was also shown to be produced through these two pathways, with Pathway II shown to be dominant in rice shoots and roots.

Mitochondria: the birth place, battle ground and the site of melatonin metabolism in cells

“…This alternative pathway for melatonin synthesis has been confirmed in plants (48). In that study, the results indicated that during normal condition, a majority of melatonin was the product of ASMT as the last enzyme; however, under stressful conditions, the major part of melatonin was generated by catalytic activity of SNAT as the final enzyme (49).…”

Effects of Light Quality and Phytochrome Form on Melatonin Biosynthesis in Rice

“…In each step, multiple isogenes participate in precursor synthesis; for this reason, it is difficult to generate melatonin knockout plants. Due to the low catalytic efficiency of the final two enzymes (SNAT and ASMT), plant melatonin levels are extremely low; therefore, precise quantification of melatonin levels remains challenging, even with the use of cutting-edge liquid chromatography-mass spectrometry (LC-MS) analytical tools [14,30]. However, plant melatonin synthesis can be induced in response to various stimuli, including pathogen infection and cadmium treatment [30,31].…”

“…Due to the low catalytic efficiency of the final two enzymes (SNAT and ASMT), plant melatonin levels are extremely low; therefore, precise quantification of melatonin levels remains challenging, even with the use of cutting-edge liquid chromatography-mass spectrometry (LC-MS) analytical tools [14,30]. However, plant melatonin synthesis can be induced in response to various stimuli, including pathogen infection and cadmium treatment [30,31]. Among many candidates, cadmium is optimal for eliciting melatonin synthesis in rice [32]; however, it cannot induce melatonin production under dark conditions [33].…”

iTRAQ-based proteomic and physiological analyses of mustard sprouts in response to heat stress

“…Melatonin, tryptophan and intermediate in rice samples increased signicantly aer treated with different concentrations of cadmium in comparison to the control. 32 In this way, the increase in the levels of four tryptophan biosynthesis-related DAPs (Q9FFW8, Q94JT5, Q42529, F4K0T5) under heat stress treatment in the present study might contribute to the enhancement of melatonin in mustard sprouts.…”