Successful expression of a novel bacterial gene for pinoresinol reductase and its effect on lignan biosynthesis in transgenic Arabidopsis thaliana
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Pinoresinol reductase and pinoresinol/lariciresinol reductase play important roles in an early step of lignan biosynthesis in plants. The activities of both enzymes have also been detected in bacteria. In this study, pinZ, which was first isolated as a gene for bacterial pinoresinol reductase, was constitutively expressed in Arabidopsis thaliana under the control of the cauliflower mosaic virus 35S promoter. Higher reductive activity toward pinoresinol was detected in the resultant transgenic plants but not in wild-type plant. Principal component analysis of data from untargeted metabolome analyses of stem, root, and leaf extracts of the wild-type and two independent transgenic lines indicate that pinZ expression caused dynamic metabolic changes in stems, but not in roots and leaves. The metabolome data also suggest that expression of pinZ influenced the metabolisms of lignan and glucosinolates but not so much of neolignans such as guaiacylglycerol-8-O-4′-feruloyl ethers. In-depth quantitative analysis by liquid chromatography–tandem mass spectrometry (LC-MS/MS) indicated that amounts of pinoresinol and its glucoside form were markedly reduced in the transgenic plant, whereas the amounts of glucoside form of secoisolariciresinol in transgenic roots, leaves, and stems increased. The detected levels of lariciresinol in the transgenic plant following β-glucosidase treatment also tended to be higher than those in the wild-type plant. Our findings indicate that overexpression of pinZ induces change in lignan compositions and has a major effect not only on lignan biosynthesis but also on biosynthesis of other primary and secondary metabolites.
KeywordsArabidopsis thaliana Lignan Pinoresinol reductase Sphingobium sp. SYK-6
The authors thank Mr. Yasuyuki Ishikawa for the preparation of a binary vector for plant transformation and the subsequent screening of transgenic lines in an earlier stage of this study. The authors also acknowledge Prof. Yoshikazu Kitano for his help in the chiral HPLC analysis and Prof. Toshiaki Umezawa for providing pinoresinol. This work was supported in part by the New Energy and Industrial Technology Development Organization (Development of Preparatory Basic Bioenergy Technology), by the Japan Science and Technology Agency (Advanced Low Carbon Technology Research and Development Program), and by the Japan Society for the Promotion of Science (Japan Advanced Plant Science Network Program).
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