Purification and identification of naringenin 7-O-methyltransferase, a key enzyme in biosynthesis of flavonoid phytoalexin sakuranetin in rice

Abstract

Sakuranetin, the major flavonoid phytoalexin in rice, is induced by ultraviolet (UV) irradiation, CuCl(2) treatment, jasmonic acid treatment, and infection by phytopathogens. It was recently demonstrated that sakuranetin has anti-inflammatory activity, anti-mutagenic activity, anti-pathogenic activities against Helicobacter pylori, Leishmania, and Trypanosoma and contributes to the maintenance of glucose homeostasis in animals. Thus, sakuranetin is a useful compound as a plant antibiotic and a potential pharmaceutical agent. Sakuranetin is biosynthesized from naringenin by naringenin 7-O-methyltransferase (NOMT). In previous research, rice NOMT (OsNOMT) was purified to apparent homogeneity from UV-treated wild-type rice leaves, but the purified protein, named OsCOMT1, exhibited caffeic acid O-methyltransferase (COMT) activity and not NOMT activity. In this study, we found that OsCOMT1 does not contribute to sakuranetin production in rice in vivo, and we purified OsNOMT using the oscomt1 mutant. A crude protein preparation from UV-treated oscomt1 leaves was subjected to three sequential purification steps, resulting in a 400-fold purification from the crude enzyme preparation. Using SDS-PAGE, the purest enzyme preparation showed a minor band at an apparent molecular mass of 40 kDa. Two O-methyltransferase-like proteins, encoded by Os04g0175900 and Os12g0240900, were identified from the 40-kDa band by MALDI-TOF/TOF analysis. Recombinant Os12g0240900 protein showed NOMT activity, but the recombinant Os04g0175900 protein did not. Os12g0240900 expression was induced by jasmonic acid treatment in rice leaves prior to sakuranetin accumulation, and the Os12g0240900 protein showed reasonable kinetic properties to OsNOMT. On the basis of these results, we conclude that Os12g0240900 encodes an OsNOMT.

FIGURE 1.

Biosynthesis of sakuranetin from naringenin.

FIGURE 2.

Genome structure and gene expression of OsCOMT1 in the oscomt1 mutant. A, tDNA sequence is inserted 374 bp downstream of the transcription initiation site of OsCOMT1 in the oscomt1 mutant. Black arrowheads (#1, #2, and #3) indicate the locations of primers used for genomic PCR, and white arrowheads indicate the locations of primers used for expression analysis. B, results of genomic PCR of the oscomt1 mutant allele. C, transcriptional expression of OsCOMT1 in wild-type rice and the oscomt1 mutant. Ubiquitin expression was used as a control.

FIGURE 3.

Accumulation of sakuranetin and NOMT and COMT enzymatic activity in elicited oscomt1 and wild-type rice leaves. A, accumulated sakuranetin in oscomt1 and wild-type rice leaves 48 h after CuCl₂ treatment was determined by LC-MS/MS. B and C, specific NOMT (B) and COMT (C) activities of crude enzyme preparations from oscomt1 and wild-type rice leaves 48 h after CuCl₂ treatment. Amounts of sakuranetin and ferulic acid extracted from the enzymatic reaction mixture were measured by LC-MS/MS, and the specific activities were determined. The data are means ± S.D. of three replicates. An asterisk indicates significant differences compared with wild-type rice in a t test at p < 0.05.

FIGURE 4.

Identification of O-methyltransferase-like proteins by MALDI-TOF/TOF analysis. A, Coomassie-stained SDS-PAGE of purified enzyme preparations is shown. Lane 1, crude enzyme; lanes 2–4, ammonium sulfate precipitation-, DEAE-, and adenosine-agarose affinity-purified enzymes; lane M, protein marker. Black arrowhead indicates the 40-kDa proteins, which include OsNOMT. White arrowheads indicate the major components of Rubisco-related proteins. B and C, amino acid sequences of O-methyltransferase-like proteins encoded by gene loci Os04g0175900 (B) and Os12g0240900 (C) obtained from the 40-kDa band by MALDI-TOF/TOF analysis are shown. Bold italic characters indicate the fragments identified by MALDI-TOF/TOF analysis.

FIGURE 5.

NOMT enzymatic activity of GST-fused recombinant proteins. Crude enzyme preparations from E. coli expressing either GST-2409 or GST-1759 were purified by affinity chromatography with GSTrap HP column, and the purified preparations were applied to SDS-PAGE and NOMT enzymatic activity assays. A, Coomassie-stained SDS-PAGE of affinity purified GST-1759 and GST-2409. (C, crude; P, purified). B, LC-MS/MS chromatograms of reaction products in the NOMT enzymatic activity assay using either GST-2409 or GST-1759 and an authentic standard of sakuranetin. C, Hanes-Woolf plots for GST-2409 with natural naringenin. The concentration of natural naringenin was calculated as the half of racemic naringenin. The data are means ± S.E. of three replicates.

FIGURE 6.

Relative methylation activities of OsNOMT against flavonoids and other phenolic compounds. Purified GST-OsNOMT was incubated with 0.3 mm of phenolic substrates and a trace amount of S-[methyl-³H]adenosyl-l-methionine. The reaction was terminated by adding 1 m HCl. The reaction products were extracted with ethyl acetate and the incorporated methyl-³H was measured. The data are means ± S.E. of three replicates. Different characters indicate significant differences in a Tukey's test (p < 0.05) among the activities against the substrates.

FIGURE 7.

Time course analysis of OsNOMT mRNA and accumulation of sakuranetin and naringenin in JA-treated wild-type rice leaves. A, relative expression levels of OsNOMT in rice leaves from 0 to 72 h after treatment with 100 μm JA. The mRNA levels were determined using qRT-PCR. Each value was normalized to the OsUBQ mRNA level. The data are means ± S.D. of three experiments. B and C, accumulated amounts of sakuranetin (B) and naringenin (C) from 0 to 72 h after treatment with 100 μm JA were quantified using LC-MS/MS. The data are means ± S.E. of three experiments.