The Antioxidant Cyclic 3-Hydroxymelatonin Promotes the Growth and Flowering of Arabidopsis thaliana

Abstract

In plants, melatonin is metabolized into several compounds, including the potent antioxidant cyclic 3-hydroxymelatonin (3-OHM). Melatonin 3-hydroxylase (M3H), a member of the 2-oxo-glutarate-dependent enzyme family, is responsible for 3-OHM biosynthesis. Although rice M3H has been cloned, its roles are unclear, and no homologs in other plant species have been characterized. Here, we cloned and characterized Arabidopsis thaliana M3H (AtM3H). The purified recombinant AtM3H exhibited K_m and V_max values of 100 μM and 20.7 nmol/min/mg protein, respectively. M3H was localized to the cytoplasm, and its expression peaked at night. Based on a 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay, 3-OHM exhibited 15-fold higher antioxidant activity than melatonin. An Arabidopsis M3H knockout mutant (m3h) produced less 3-OHM than the wildtype (WT), thus reducing antioxidant activity and biomass and delaying flowering. These defects were caused by reduced expression of FLOWERING LOCUS T (FT) and gibberellin-related genes, which are responsible for flowering and growth. Exogenous 3-OHM, but not exogenous melatonin, induced FT expression. The peak of M3H expression at night matched the FT expression pattern. The WT and m3h exhibited similar responses to salt stress and pathogens. Collectively, our findings indicate that 3-OHM promotes growth and flowering in Arabidopsis.

Keywords: Escherichia coli expression; FLOWERING LOCUS T; antioxidant; cyclic 3-hydroxymelatonin; gibberellin; melatonin; pathogen resistance; salt tolerance.

Figure 1

Reaction catalyzed by M3H and amino acid sequences of rice OsM3H and Arabidopsis AtM3H. (A) Enzymatic conversion of M2H and M3H. (B) Amino acid sequences of OsM3H and AtM3H. Asterisks, identical amino acids; dashes, gaps; underlining, conserved 2-ODD domain. GenBank accession numbers of OsM3H and AtM3H, AK067086 and AT1g17020, respectively.

Figure 2

Affinity purification of His-tagged Arabidopsis AtM3H and M3H and M2H activities. (A) Expression and affinity purification of His-tagged AtM3H from E. coli. (B) Specific activities of M3H and M2H. Proteins were resolved by SDS-PAGE followed by Coomassie blue staining. M, molecular standard; lane 1, total protein in 10 µL aliquots of bacterial suspension without IPTG; lane 2, total protein after IPTG induction; lane 3, 10 µg of protein in the supernatant after centrifugation at 10,000× g; lane 4, AtM3H protein (10 µg) purified by affinity (Ni-NTA) chromatography.

Figure 3

HPLC of in vitro products of purified recombinant AtM3H. (A) 2-OHM authentic compound. (B) In vitro product of AtM3H. (C) 3-OHM authentic compound. (D) In vitro product of AtM3H. EU, emission units; AU, absorption units.

Figure 4

Kinetics of purified AtM3H. M3H activity as a function of (A) pH and (B) temperature. (C) Effects of cofactors on M3H activity. (D) Effect of prohexadione-Ca on M3H activity. Means ± SD of three replicates. Different letters (a–d) indicate significant differences at p < 0.05. PC, M3H activity with all cofactors; α-KG, α-ketoglutarate; Asc, ascorbic acid; nd, not detected.

Figure 5

Enzyme kinetic analysis. V_max and K_m based on Lineweaver–Burk plots.

Figure 6

Subcellular localization. (A) Red fluorescence of AtM3H-mCherry. (B) Green fluorescence of cytoplasmic GFP-HA. (C) Merged fluorescence image of A and B. Tobacco leaves were infiltrated with Agrobacterium tumefaciens (GV2260) containing XVE-inducible AtM3H-mCherry or constitutive 35S promoter-driven GFP-HA (cytoplasm marker). Bars, 20 μm.

Figure 7

Schematic diagram of the genome structure and antioxidant activity of m3h. (A) Antioxidant activities of melatonin, 2-OHM, and 3-OHM revealed by DPPH assay. (B) Schematic of the T-DNA insertion site (downward arrow) in M3H. Gray and white boxes indicate exons and introns, respectively (not to scale). (C) Expression of M3H in the WT and m3h revealed by RT-PCR over 24 h. Numbers in parentheses are numbers of PCR cycles. Elongation factor-1 alpha (EF-1α) (AT5g60390) served as the loading control. (D) 3-OHM levels in leaves of the WT and m3h infiltrated with melatonin (100 µM). (E) Antioxidant activities of lower leaves of 30-day-old WT and m3h, as revealed by DPPH assay. Error bars are standard deviations of three biological replicates. Different letters and asterisks (*) indicate significant differences (Tukey’s post hoc HSD test; p < 0.05). RSA, radical scavenging activity.

Figure 8

Phenotypic characterization of m3h. (A) Representative flowering phenotypes. (B) Rosette leaf phenotypes. (C) Flowering times of the WT and m3h, measured as the number of rosette leaves at bolting. (D) Time course of plant height after bolting of the WT and m3h. (E) Leaf area measurements by leaf position. (F) Fresh weight of the WT and m3h at bolting. Arabidopsis plants were grown under a 14 h light/10 h dark cycle and photographed 4–6 days after bolting (39 days old). Scale bars, 2 cm. Error bars are standard deviations of three biological replicates. Asterisks (*) indicate significant differences (Tukey’s post hoc HSD test; p < 0.05).

Figure 9

Expression profiles of flowering-related genes in the WT and m3h. (A) Daily expression of FLOWERING LOCUS T (FT) and gibberellin (GA)-related genes in the WT and m3h. (B) Expression levels of FT, KS, and MYB33 in WT leaves in response to melatonin and 3-OHM (4 μM). Transcript levels were analyzed by RT-PCR using cDNA from leaves of 30-day-old mature plants before the bolting stage. Plants were sampled at the indicated times. EF-1α was used as the loading control. Numbers in parentheses are numbers of PCR cycles. Effector solutions were infiltrated into the abaxial leaf side at 8 pm and incubated for 2 and 4 h in darkness. Accession numbers: FT, AT1G65480; KS, AT1g15550; GA3ox1, AT1g80340; GA3ox2, AT5g06100; MYB33, AT5g60390; and EF-1α, AT5g60390.

Figure 10

Stress responses of the WT and m3h. (A) Root length in response to salt stress. Seeds were sown on MS medium in the absence (−) and presence (+) of 100 mM NaCl and vertically positioned for 3 weeks. (B) Bacterial growth in response to virulent P. syringae pv. tomato DC3000 (Pst). (C) Bacterial growth in response to avirulent P. syringae pv. tomato DC3000 containing avrRpm1 (Pst-Rpm1). Bacteria were spray-inoculated at 5 × 10⁶ (Pst) or 1 × 10⁸ CFU/mL (Pst-Rpm1) (in 0.01% [v/v] Silwet L-77), and their growth was determined at 5 days post-infection. Experiments were conducted in triplicate. Different letters indicate significant differences (Tukey’s post hoc HSD test; p < 0.05). CFU, colony-forming units.