Functional Characterization of the Ciliate Stylonychia lemnae Serotonin N-Acetyltransferase, a Pivotal Enzyme in Melatonin Biosynthesis and Its Overexpression Leads to Peroxidizing Herbicide Tolerance in Rice

Serotonin <i>N</i>-acetyltransferase (SNAT) is a pivotal enzyme for melatonin biosynthesis in all living organisms. It catalyzes the conversion of serotonin to <i>N</i>-acetylserotonin (NAS) or 5-methoxytrypytamine (5-MT) to melatonin. In contrast to animal- and plant-specific <i>SNAT</i> genes, a novel clade of archaeal <i>SNAT</i> genes has recently been reported. In this study, we identified homologues of archaeal <i>SNAT</i> genes in ciliates and dinoflagellates, but no animal- or plant-specific <i>SNAT</i> homologues. Archaeal <i>SNAT</i> homologue from the ciliate <i>Stylonychia lemnae</i> was annotated as a putative <i>N</i>-acetyltransferase. To determine whether the putative <i>S. lemnae SNAT</i> (<i>SlSNAT</i>) exhibits SNAT enzyme activity, we chemically synthesized and expressed the full-length <i>SlSNAT</i> coding sequence (CDS) in <i>Escherichia coli</i>, from which the recombinant SlSNAT protein was purified by Ni²⁺ affinity column chromatography. The recombinant SlSNAT exhibited SNAT enzyme activity toward serotonin (<i>K</i>_m = 776 µM) and 5-MT (<i>K</i>_m = 246 µM) as substrates. Furthermore, <i>SlSNAT</i>-overexpressing (SlSNAT-OE) transgenic rice plants showed higher levels of melatonin synthesis than wild-type controls. The SlSNAT-OE rice plants exhibited delayed leaf senescence and tolerance against treatment with the reactive oxygen species (ROS)-inducing herbicide butafenacil by decreasing hydrogen peroxide (H₂O₂) and malondialdehyde (MDA) levels, suggesting that melatonin alleviates ROS production in vivo.