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

Abstract

Serotonin N-acetyltransferase (SNAT) is a pivotal enzyme for melatonin biosynthesis in all living organisms. It catalyzes the conversion of serotonin to N-acetylserotonin (NAS) or 5-methoxytrypytamine (5-MT) to melatonin. In contrast to animal- and plant-specific SNAT genes, a novel clade of archaeal SNAT genes has recently been reported. In this study, we identified homologues of archaeal SNAT genes in ciliates and dinoflagellates, but no animal- or plant-specific SNAT homologues. Archaeal SNAT homologue from the ciliate Stylonychia lemnae was annotated as a putative N-acetyltransferase. To determine whether the putative S. lemnae SNAT (SlSNAT) exhibits SNAT enzyme activity, we chemically synthesized and expressed the full-length SlSNAT coding sequence (CDS) in Escherichia coli, from which the recombinant SlSNAT protein was purified by Ni²⁺ affinity column chromatography. The recombinant SlSNAT exhibited SNAT enzyme activity toward serotonin (K_m = 776 µM) and 5-MT (K_m = 246 µM) as substrates. Furthermore, SlSNAT-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.

Keywords: Osl20; Stylonichia lemnae; archaea; ciliophoran; melatonin; serotonin N-acetyltransferase; transgenic rice.

Figure 1

(A) Phylogenetic tree of Stylonichia lemnae SNAT and archaeal ortholog genes. The scale bar represents 0.4 substitutions per site. S. lemnae SNAT is written in bold for emphasis. (B) Amino acid sequence identity and similarity between S. lemnae SNAT and human Naa50 (SNAT). The conserved acetyl-coenzyme-A-binding sites are underlined. Dashes denote gaps. GenBank accession numbers are archaea SNAT (NC_002689), E. coli RimI (WP_137442509), human Naa50 (BAB14397), rice SNAT3 (AK241100), and S. lemnae SNAT (CDW73552).

Figure 2

(A) Nucleotide alignment between native (red; CDW73552) and synthetic (blue) S. lemnae SNAT. Identity is denoted by stars. Black letters, amino acids. (B) Modification of S. lemnae SNAT codons. The nucleotide sequence of synthetic S. lemnae SNAT was manually codon optimized with reference to the rice SNAT2 codon.

Figure 3

Escherichia coli expression, affinity purification of SlSNAT recombinant protein, and its enzymatic characteristics. (A) Expression of SlSNAT as a thioredoxin (Trx) fusion protein using a pET32b vector and expression of SlSNAT as an N-terminal His × 6-tagged SlSNAT protein using a pET300 vector. (B) Serotonin N-acetyltransferase enzyme activity (SNAT) as a function of various substrates. The expression of recombinant SlSNAT protein is marked by arrows.

Figure 4

SNAT enzyme kinetic analysis. Serotonin N-acetyltransferase enzyme activity as a function of (A) various temperature, (B) various pH, (C) K_m and V_max values for serotonin substrate, (D) K_m and V_max values for 5-methoxytryptamine (5-MT) substrate. Values are means ± SD (n = 3). nd, not detectable.

Figure 5

Generation of SlSNAT overexpression transgenic rice and the melatonin content of rice seedlings. (A) RT-PCR analyses of transgenic and wild-type 7-day-old rice seedlings. (B) Melatonin contents of 7-day-old rice seedlings. (C) Photograph of seed length. (D) Photograph of lamina angle in 3-week-old rice seedling. (E) Measurement of lamina angle. WT, wild type; UBQ5, rice ubiquitin 5 gene. GenBank accession number of UBQ5 is AK061988. Different letters indicate significant differences among lines (Tukey’s HSD; p < 0.05).

Figure 6

Enhanced senescence tolerance in SlSNAT-overexpressing transgenic rice plants. (A) Photograph of senescence-treated 5-week-old rice leaves. (B) Chlorophyll contents in senescence-treated rice leaves. (C) Malondialdehyde (MDA) contents. (D) Gene expression profiles of senescence marker genes by RT-PCR. (E) Gene expression profiles of senescence marker genes by quantitative RT-PCR. Fourth and fifth leaves from 5-week-old rice plants grown in soil pots were detached and this was followed by senescence treatment for 12 days. WT, wild type; UBQ5, rice ubiquitin 5 gene. GenBank accession numbers are Osl2 (AF251073), Osl20 (AF251067), Osl185 (AF251075), and UBQ5 (AK061988). Different letters indicate significant differences among the lines (Tukey’s HSD; p < 0.05).

Figure 7

Enhanced tolerance of SlSNAT-overexpressing transgenic rice plants against peroxidizing herbicide butafenacil. (A) Photograph of rice seedlings after butafenacil treatment. (B) Effect of butafenacil treatment on cellular leakage. (C) MDA production from butafenacil-treated rice seedlings. (D) H₂O₂ content from butafenacil-treated rice seedlings. Seven-day-old rice seedlings were challenged with 0.1 µM butafenacil for 48 h. WT, wild type; FW, fresh weight. Different letters indicate significant differences among the lines (Tukey’s HSD; p < 0.05).

Figure 8

Sequence comparison and phylogenetic tree of SNAT in the Ciliophora and dinoflagellates. (A) Consensus amino acid sequences among three SNAT proteins including the human Naa50, the ciliate Stylonichia lemnae SNAT, and the dinoflagellate Polarella glacialis SNAT. Bold red letters indicate consensus amino acids. Dashes denote gaps for maximizing alignment of conserved residues. A coenzyme-A-binding pocket is underlined. (B) Phylogenetic tree analysis of SNAT proteins from the ciliates and dinoflagellates. GenBank accession numbers of various SNAT genes are as follows: human Naa50 (BAB14397), Cladocopium goreaui (CAI3999280); Effrenium voratum (CAJ1361560); Polarella glacialis (CAK0876941); Stylonichia lemna (CCKQ01002460); Paramecium sonneborni (CAD8056267); Pseudocohnilembus persalius (KRX00195); Tetrahymena thermophila SB210 (XP_001025216); Ichthyophthirius multifiliis (XP-004035125). The scale bar represents 0.3 substitutions per site.