Melatonin biosynthesis requires N-acetylserotonin methyltransferase activity of caffeic acid O-methyltransferase in rice

Abstract

Caffeic acid O-methyltransferase (COMT) methylates N-acetylserotonin into melatonin; that is, it has N-acetylserotonin O-methyltransferase (ASMT) activity. The ASMT activity of COMT was first detected in Arabidopsis thaliana COMT (AtCOMT). To confirm the involvement of COMT on melatonin synthesis in other plant species, the ASMT activity of a COMT from rice (Oryza sativa) (OsCOMT) was evaluated. Purified recombinant OsCOMT protein from Escherichia coli was used to validate the high ASMT activity of OsCOMT, similar to that of AtCOMT. The K m and V max values for the ASMT activity of OsCOMT were 243 µM and 2400 pmol min(-1) mg protein(-1), which were similar to those of AtCOMT. Similar to AtCOMT, OsCOMT was localized in the cytoplasm. In vitro ASMT activity was significantly inhibited by either caffeic acid or quercetin in a dose-dependent manner. Analogously, in vivo production of melatonin was significantly inhibited by quercetin in 4-week-old detached rice leaves. Lastly, the transgenic rice plants overexpressing rice COMT showed an increase in melatonin levels whereas transgenic rice plants suppressing the rice COMT had a significant decrease on melatonin levels, suggestive of the direct role of COMT in melatonin biosynthesis in plants.

Keywords: N-Acetylserotonin O-methyltransferase; caffeic acid O-methyltransferase; melatonin; transgenic rice..

Fig. 1.

Amino acid sequence comparisons between AtCOMT and OsCOMT. The SAM-binding sites are underlined and the phenolic substrate-binding sites shaded. The three catalytic residues of COMT (His267, Glu295, and Glu327) are shown in bold letters.

Fig. 2.

Purification of recombinant OsCOMT protein and enzyme activity. (A) Purification of N-terminal 6× histidine-tagged OsCOMT. ASMT activity of purified OsCOMT according to (B) enzyme concentration and (C) temperature. E. coli harbouring pET300-OsCOMT was incubated with 1mM IPTG (isopropyl-β-d-thiogalactopyranoside) at 28 °C for 5h. Protein samples were separated by SDS-PAGE and stained with Coomassie blue. M, molecular mass standards; lane 1, total proteins in 15-µl aliquots of bacterial culture without IPTG; lane 2, total proteins in 15-µl aliquots of bacterial culture with IPTG; lane 3, 20 µg soluble protein; lane 4, 10 µg OsCOMT purified by affinity chromatography. In vitro melatonin production was measured at 37 °C or varying temperatures using purified N-terminal 6× histidine-tagged OsCOMT. These data represent the mean ± standard deviation of triplicate experiments.

Fig. 3.

Effects of various substrates on ASMT activity. (A) Inhibition of ASMT activity by caffeic acid and (B) quercetin. ASMT activity assays were performed in the presence of various concentrations of either caffeic acid or quercetin. ASMT activity assays for OsCOMT were performed in the presence of 0.5mM NAS and various concentrations of caffeic acid or quercetin. Asterisks (*) indicate a significant difference from the control (P<0.05).

Fig. 4.

Measurement of the K _m and V _max of OsCOMT for NAS. OsCOMT (1 µg) was incubated with different concentrations of substrate for 30min at 37 °C. The K _m and V _max values were determined using Lineweaver–Burk plots.

Fig. 5.

Localization of OsCOMT. (A) Red fluorescence of OsCOMT-mCherry and (B) chlorophyll (Chl) autofluorescence. (C) The two fluorescence images were merged (A+B). Tobacco (N. benthamiana) leaves were infiltrated with Agrobacterium harbouring the XVE-inducible OsCOMT-mCherry binary vector, as described in Materials and methods. Bars, 40 μm. (This figure is available in colour at JXB online.)

Fig. 6.

Melatonin quantification in the detached rice leaves upon cadmium treatment. (A) Proposed melatonin biosynthesis in rice via the COMT enzyme, which is inhibited by quercetin. (B) Melatonin contents in response to caffeic acid or quercetin. The detached leaves of 4-week-old rice plants were transferred into a 50-ml polypropylene conical tube containing 15ml water with either 0.1mM caffeic acid or 0.1mM quercetin and incubated for 1 d. The samples were then treated with 0.2mM cadmium for inducing melatonin synthesis for 3 d at 28 °C under a 16-h light/8-h dark cycle. The data represent the means ± standard errors of three replicates. FW: fresh weight. Asterisks (*) indicate a significant difference from the control (P<0.05).

Fig. 7.

Binary vector structure and generation of COMT overexpression and suppression transgenic rice plants. (A) Schematic diagram of binary vectors of pIPKb002:OsCOMT and pTCK303:OsCOMT. (B) Flag leaves (T₀) used for total RNA isolation and RT-PCR analysis of wild-type and transgenic lines. Eight independent transgenic lines were generated and grown in a paddy field. The numbers in parentheses indicate the number of PCR cycles. Ubi-P, maize ubiquitin promoter; Tnos, nopaline synthase terminator; HPT, hygromycin phosphotransferase; WT, wild-type. UBQ5, rice ubiquitin5 gene.

Fig. 8.

Expression levels of COMT transcript and ASMT enzyme activity. (A) RT-PCR analysis of COMT mRNA in wild-type and transgenic lines. (B) ASMT enzyme activity in the shoot of wild-type and transgenic lines. (C) ASMT enzyme activity in the root of wild-type and transgenic lines. Seven-day-old seedlings grown in a half strength MS medium were employed. The numbers in parentheses indicate the number of PCR cycles used. Asterisks (*) indicate significant differences from the wild-type (P<0.05). WT, wild-type; OX lines, COMT-overexpressed transgenic lines (T2); RNAi lines, COMT-suppressed RNAi transgenic lines (T2).

Fig. 9.

COMT enzyme activity and melatonin levels in wild-type and transgenic lines (T2). (A) COMT enzyme activity measurements in wild-type and transgenic lines. (B) Melatonin levels in wild-type and transgenic lines. Shoot of 7-d old seedlings were employed for COMT enzyme activity. As for melatonin analysis, 1-month-old rice leaves were detached and challenged with 0.2mM cadmium for 3 d. Asterisks (*) indicate significant differences from the wild-type (P<0.05). WT, wild-type; OX lines, COMT-overexpressed transgenic lines; RNAi lines, COMT-suppressed RNAi transgenic lines.