Cadmium‐induced melatonin synthesis in rice requires light, hydrogen peroxide, and nitric oxide: Key regulatory roles for tryptophan decarboxylase and caffeic acid O‐methyltransferase

Abstract

In plants, melatonin production is induced by stimuli such as cold and drought, and cadmium (Cd) is the best elicitor of melatonin production in rice. However, the mechanism by which Cd induces melatonin synthesis in plants remains unknown. We challenged rice seedlings with Cd under different light conditions and found that continuous light produced the highest levels of melatonin, while continuous dark failed to induce melatonin production. Transcriptional and translational induction of tryptophan decarboxylase contributed to the light induction of melatonin during Cd treatment, whereas the protein level of light-induced caffeic acid O-methyltransferase (COMT) was decreased by Cd treatment. In analogy, COMT enzyme activity was inhibited in vitro by Cd in a dose-dependent manner. Notably, the Cd-induced melatonin synthesis was significantly impaired by treatment with either an H O production inhibitor (DPI) or an NO scavenger (cPTIO). The combination of both inhibitors almost completely abolished Cd-induced melatonin synthesis, suggesting an absolute requirement for H O and NO. However, neither serotonin nor N-acetylserotonin (NAS) was induced by H O alone. In contrast, NO significantly induced serotonin production but not NAS or melatonin production. This indicated that serotonin did not enter chloroplasts, where serotonin N-acetyltransferase (SNAT) is constitutively expressed. This suggests that chloroplastidic SNAT expression prevents increased melatonin production after exposure to stress, ultimately leading to the maintenance of a steady-state melatonin level inside cells.

Melatonin is involved in skotomorphogenesis by regulating brassinosteroid biosynthesis in rice plants

“…The resulting T 1 and T 2 seeds were selected in the presence of the antibiotic hygromycin. These hygromycin‐resistant seedlings (T 1 ) were grown for 4 weeks in soil in a glasshouse, and detached leaves were subjected to 0.2 mmol/L cadmium for 3 days under 12‐hour light/12‐hour dark cycle to induce melatonin synthesis . As expected, all transgenic SNAT2 RNAi lines produced lower levels of melatonin than the wild type (Figure C).…”

“…Of note, transcripts of the 2 SNAT1 and SNAT2 isogenes were downregulated in response to melatonin treatment. This sort of feedback inhibition on SNAT transcripts has been reported when rice plants are challenged with cadmium, a potent elicitor of melatonin induction in plants …”

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

“…Seven‐day‐old rice seedlings were subjected to cadmium treatment. In brief, a group of 10 seedlings was incubated in a 50‐mL conical tube in water containing 0.5 mmol/L CdCl 2 (Sigma‐Aldrich, St. Louis, MO) at 28°C for 3 days under continuous light . The leaves and stems were harvested for RNA extraction and further chemical analyses.…”

“…Frozen rice seedlings (0.1 g samples) were ground into powder in liquid nitrogen using the TissueLyser II (Qiagen) and extracted with 1 mL of chloroform for 1 hour at room temperature. The chloroform extracts (200 µL) were completely evaporated and dissolved in 0.1 mL of 40% methanol, and 10 µL aliquots was subjected to high‐performance liquid chromatography (HPLC) using a fluorescence detector system (Waters, Milford, MA), as described previously . Serotonin and N ‐acetylserotonin were quantified as described previously using the HPLC system .…”

Flavonoids inhibit both rice and sheep serotonin N‐acetyltransferases and reduce melatonin levels in plants

“…The fact that plant melatonin levels are low is well supported by recent analyses of melatonin biosynthetic gene expression and the cloning of several melatonin catabolic genes including melatonin 2‐hydroxylase and melatonin 3‐hydroxylase . The chloroplast SNAT of plants may limit melatonin production in adverse environments and in the presence of nitric oxide . Unlike animals, plants synthesize melatonin in 3 distinct subcellular organelles using several pathways .…”