Dataset Information

Identification of a plastid-localized bifunctional nerolidol/linalool synthase in relation to linalool biosynthesis in young grape berries.

ABSTRACT: Monoterpenoids are a diverse class of natural products and contribute to the important varietal aroma of certain Vitis vinifera grape cultivars. Among the typical monoterpenoids, linalool exists in almost all grape varieties. A gene coding for a nerolidol/linalool (NES/LINS) synthase was evaluated in the role of linalool biosynthesis in grape berries. Enzyme activity assay of this recombinant protein revealed that it could convert geranyl diphosphate and farnesyl diphosphate into linalool and nerolidol in vitro, respectively, and thus it was named VvRILinNer. However, localization experiment showed that this enzyme was only localized to chloroplasts, which indicates that VvRILinNer functions in the linalool production in vivo. The patterns of gene expression and linalool accumulation were analyzed in the berries of three grape cultivars ("Riesling", "Cabernet Sauvignon", "Gewurztraminer") with significantly different levels of monoterpenoids. The VvRILinNer was considered to be mainly responsible for the synthesis of linalool at the early developmental stage. This finding has provided us with new knowledge to uncover the complex monoterpene biosynthesis in grapes.

SUBMITTER: Zhu BQ

PROVIDER: S-EPMC4284690 | biostudies-literature | 2014 Dec

REPOSITORIES: biostudies-literature

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Identification of a plastid-localized bifunctional nerolidol/linalool synthase in relation to linalool biosynthesis in young grape berries.

Zhu Bao-Qing BQ Cai Jian J Wang Zhi-Qun ZQ Xu Xiao-Qing XQ Duan Chang-Qing CQ Pan Qiu-Hong QH

International journal of molecular sciences 20141201 12

Monoterpenoids are a diverse class of natural products and contribute to the important varietal aroma of certain Vitis vinifera grape cultivars. Among the typical monoterpenoids, linalool exists in almost all grape varieties. A gene coding for a nerolidol/linalool (NES/LINS) synthase was evaluated in the role of linalool biosynthesis in grape berries. Enzyme activity assay of this recombinant protein revealed that it could convert geranyl diphosphate and farnesyl diphosphate into linalool and ne ...[more]

PMID: 25470020

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Project description:Linalool and nerolidol are terpene alcohols that occur naturally in many aromatic plants and are commonly used in food and cosmetic industries as flavors and fragrances. In plants, linalool and nerolidol are biosynthesized as a result of respective linalool synthase and nerolidol synthase, or a single linalool/nerolidol synthase. In our previous work, we have isolated a linalool/nerolidol synthase (designated as PamTps1) from a local herbal plant, Plectranthus amboinicus, and successfully demonstrated the production of linalool and nerolidol in an Escherichia coli system. In this work, the biochemical properties of PamTps1 were analyzed, and its 3D homology model with the docking positions of its substrates, geranyl pyrophosphate (C10) and farnesyl pyrophosphate (C15) in the active site were constructed. PamTps1 exhibited the highest enzymatic activity at an optimal pH and temperature of 6.5 and 30 °C, respectively, and in the presence of 20 mM magnesium as a cofactor. The Michaelis-Menten constant (Km) and catalytic efficiency (kcat/Km) values of 16.72 ± 1.32 µM and 9.57 × 10-3 µM-1 s-1, respectively, showed that PamTps1 had a higher binding affinity and specificity for GPP instead of FPP as expected for a monoterpene synthase. The PamTps1 exhibits feature of a class I terpene synthase fold that made up of α-helices architecture with N-terminal domain and catalytic C-terminal domain. Nine aromatic residues (W268, Y272, Y299, F371, Y378, Y379, F447, Y517 and Y523) outlined the hydrophobic walls of the active site cavity, whilst residues from the RRx8W motif, RxR motif, H-α1 and J-K loops formed the active site lid that shielded the highly reactive carbocationic intermediates from the solvents. The dual substrates use by PamTps1 was hypothesized to be possible due to the architecture and residues lining the catalytic site that can accommodate larger substrate (FPP) as demonstrated by the protein modelling and docking analysis. This model serves as a first glimpse into the structural insights of the PamTps1 catalytic active site as a multi-substrate linalool/nerolidol synthase.

Project description:The plant volatile linalool plays important roles in the regulation of defense responses in rice. To clarify the response to linalool in rice at gene expression level, we performed a microarray analysis using the Agilent Rice Oligo Microarray (44k, custom-made; Agilent Technologies, Redwood City, CA, USA). As a result, many defense-related genes including pathogenesis-related (PR) genes were upregulated in the linalool synthase-overexpressing transgenic rice. An Agilent Rice Oligo Microarray (44k, custom-made; Agilent Technologies, Redwood City, CA, USA) was used for the microarray analysis. Total RNA was extracted from leaf blades of four-leaf stage rice plants. Line 13 was used as the linalool synthase-overexpressing transgenic rice plant. For each biological replicate, material from at least three rice plants was pooled to provide a single sample for RNA extraction. We performed 3 biological replicates for each treatment. All microarray procedures and data analyses were performed according to the manufacturerM-bM-^@M-^Ys instructions. RNA integrity was checked using an Agilent 2100 Bioanalyzer (Agilent Technologies, Redwood City, CA, USA). Total RNA (100 ng) was labeled with Cy-3 using an Agilent Low Input Quick Amp Labeling Kit, One-Color (Agilent Technologies). Fluorescently labeled targets were hybridized to Agilent Rice Oligo Microarrays for 17 h at 65M-BM-0C. After hybridization, following wash processes were performed according to the manufacturerM-bM-^@M-^Ys instructions, and hybridized microarrays were scanned using an Agilent Microarray Scanner (G2565CA; Agilent Technologies). Feature extraction software (Agilent Feature Extraction; Agilent Technologies) was used to delineate and measure the signal intensity of each spot in the array, and to normalize intensities. The background was measured around each spot as local background, calculated by the Feature Extraction software. Statistical data extraction processes were performed according to the manufacturerM-bM-^@M-^Ys instructions.

Dataset Information

Identification of a plastid-localized bifunctional nerolidol/linalool synthase in relation to linalool biosynthesis in young grape berries.

Publications

Identification of a plastid-localized bifunctional nerolidol/linalool synthase in relation to linalool biosynthesis in young grape berries.

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