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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.

SUBMITTER: Shimizu T

PROVIDER: S-EPMC3365963 | biostudies-literature | 2012 Jun

REPOSITORIES: biostudies-literature

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

Shimizu Takafumi T Lin Fengqiu F Hasegawa Morifumi M Okada Kazunori K Nojiri Hideaki H Yamane Hisakazu H

The Journal of biological chemistry 20120409 23

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 p ...[more]

PMID: 22493492

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Project description:Sakuranetin, the major flavonoid phytoalexin in rice, can be induced by ultraviolet (UV) irradiation, treatment with CuCl 2 or jasmonic acid (JA), or phytopathogenic infection. In addition to sakuranetin's biological significance on disease resistance in rice, its broad bioactivities have recently been described. Results from these studies have shown that sakuranetin is a useful compound as a plant antibiotic and a potential pharmaceutical agent. Sakuranetin is biosynthesized from naringenin, a precursor of sakuranetin, by naringenin 7-O-methyltransferase (NOMT), but the relevant gene has not yet been identified in rice. Recently, we identified the OsNOMT gene, which is involved in the final step of sakuranetin biosynthesis in rice. In previous studies, OsNOMT was purified to apparent homogeneity from UV-treated wild-type rice leaves; however, the purified protein, termed OsCOMT1, exhibited caffeic acid 3-O-methyltransferase (COMT) activity, but not NOMT activity. Based on the analysis of an oscomt1 T-DNA tagged mutant, we determined that OsCOMT1 did not contribute to sakuranetin production in rice in vivo. Therefore, we took advantage of the oscomt1 mutant to purify OsNOMT. 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 with a minor band at an apparent molecular mass of 40 kDa in the purest enzyme preparation. Matrix-assisted laser desorption/ionization time of flight/time of flight analysis showed that the 40 kDa protein band included two O-methyltransferase-like proteins, but one of the proteins encoded by Os12g0240900 exhibited clear NOMT activity; thus, this gene was designated OsNOMT. Gene expression was induced by treatment with jasmonic acid in rice leaves prior to sakuranetin accumulation, and the recombinant protein showed reasonable kinetic properties to NOMT. Identification of the OsNOMT gene enables the production of large amounts of sakuranetin through transgenic rice and microorganisms. This finding also allows for the generation of disease-resistant and sakuranetin biofortified rice in the future.

Project description:Background and aimsMicroRNAs (miRNAs) are small non-coding RNAs that act as post-transcriptional regulators of gene expression via sequence-specific cleavage or translational repression of target transcripts. They are transcribed as long single-stranded RNA precursors with unique stem-loop structures that are processed by a DICER-Like (DCL) ribonuclease, typically DCL1, to produce mature miRNAs. Although a plethora of miRNAs have been found to be regulated by pathogen infection in plants, the biological function of most miRNAs remains largely unknown. Here, the contribution of OsDCL1 to rice immunity was investigated.MethodsActivation-tagged Osdcl1a (Osdcl1a-Ac) rice mutants were examined for resistance to pathogen infection. mRNA and small RNA deep sequencing, quantitative real-time PCR (RT-qPCR) and stem-loop reverse tanscripion-PCR (RT-PCR) were used to examine DCL1a-mediated alterations in the rice transcriptome. Rice diterpene phytoalexins were quantified by liquid chromatography-tandem mass spectrometry (LC-MSMS). Accumulation of O2·- was determined by nitroblue tetrazolium (NBT) staining.Key resultsdcl1a-Ac mutants exhibit enhanced susceptibility to infection by fungal pathogens which was associated with a weaker induction of defence gene expression. Comparison of the mRNA and miRNA transcriptomes of dcl1a-Ac and wild-type plants revealed misregulation of genes involved in detoxification of reactive oxygen species. Consequently, dcl1a-Ac plants accumulated O2·- in their leaves and were more sensitive to methyl viologen-induced oxidative stress. Furthermore, dcl1a-Ac plants showed downregulation of diterpenoid phytoalexin biosynthetic genes, these genes also being weakly induced during pathogen infection. Upon pathogen challenge, dcl1a-Ac plants failed to accumulate major diterpenoid phytoalexins. OsDCL1a activation resulted in marked alterations in the rice miRNAome, including both upregulation and downregulation of miRNAs.ConclusionsOsDCL1a activation enhances susceptibility to infection by fungal pathogens in rice. Activation of OsDCL1a represses the pathogen-inducible host defence response and negatively regulates diterpenoid phytoalexin production. These findings provide a basis to understand the molecular mechanisms through which OsDCL1a mediates rice immunity.

Project description:Flavonoids are a class of key polyphenolic secondary metabolites with broad functions in plants, including stress defense, growth, development and reproduction. Oryza sativa L. (rice) is a well-known model plant for monocots, with a wide range of flavonoids, but the key flavonoid biosynthesis-related genes and their molecular features in rice have not been comprehensively and systematically characterized. Here, we identified 85 key structural gene candidates associated with flavonoid biosynthesis in the rice genome. They belong to 13 families potentially encoding chalcone synthase (CHS), chalcone isomerase (CHI), flavanone 3-hydroxylase (F3H), flavonol synthase (FLS), leucoanthocyanidin dioxygenase (LDOX), anthocyanidin synthase (ANS), flavone synthase II (FNSII), flavanone 2-hydroxylase (F2H), flavonoid 3'-hydroxylase (F3'H), flavonoid 3',5'-hydroxylase (F3'5'H), dihydroflavonol 4-reductase (DFR), anthocyanidin reductase (ANR) and leucoanthocyanidin reductase (LAR). Through structural features, motif analyses and phylogenetic relationships, these gene families were further grouped into five distinct lineages and were examined for conservation and divergence. Subsequently, 22 duplication events were identified out of a total of 85 genes, among which seven pairs were derived from segmental duplication events and 15 pairs were from tandem duplications, demonstrating that segmental and tandem duplication events play important roles in the expansion of key flavonoid biosynthesis-related genes in rice. Furthermore, these 85 genes showed spatial and temporal regulation in a tissue-specific manner and differentially responded to abiotic stress (including six hormones and cold and salt treatments). RNA-Seq, microarray analysis and qRT-PCR indicated that these genes might be involved in abiotic stress response, plant growth and development. Our results provide a valuable basis for further functional analysis of the genes involved in the flavonoid biosynthesis pathway in rice.

Dataset Information

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

Publications

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

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