Carrot (Daucus carota subsp. sativus) is a widely cultivated root vegetable of high economic importance. The aroma of carrot roots and aboveground organs is mainly defined by terpenes. We found that leaves of orange carrot cultivar also produce considerable amounts of the phenylpropenes methyleugenol and methylisoeugenol. Notably, methyleugenol is most abundant in young leaves, while methylisoeugenol is the dominant phenylpropene in mature leaf tissue. The goal of the present study was to shed light on the biochemistry and molecular biology of these compounds' biosynthesis and accumulation. Using the available genomic and transcriptomic data, we isolated a cDNA encoding eugenol/isoeugenol synthase (DcE(I)GS1), an NADPH-dependent enzyme that converts coniferyl acetate to eugenol. This enzyme exhibits dual product specificity and yields propenylphenol isoeugenol alongside allylphenol eugenol. Furthermore, we identified a cDNA encoding S-adenosyl-L-methionine:eugenol/isoeugenol O-methyltransferase 1 (DcE(I)OMT1) that produces methyleugenol and methylisoeugenol via methylation of the para-OH-group of their respective precursors. Both DcE(I)GS1 and DcE(I)OMT1 were expressed in seeds, roots, young and mature leaves, and the DcE(I)OMT1 transcript levels were the highest in leaves. The DcE(I)GS1 protein is 67% identical to anise t-anol/isoeugenol synthase and displays an apparent Km of 247 μM for coniferyl acetate. The catalytic efficiency of DcEOMT1 with eugenol is more than five-fold higher than that with isoeugenol, with Km values of 40 μM for eugenol, and of 115 μM for isoeugenol. This work expands the current knowledge of the enzymes involved in phenylpropene biosynthesis and would enable studies into structural elements defining the regioselectivity of phenylpropene synthases.

中文翻译：

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胡萝卜叶中甲基丁香酚和甲基异丁香酚的生物合成：丁香酚/异丁香酚合酶和 O-甲基转移酶的表征

胡萝卜 (Daucus carota subsp. sativus) 是一种广泛种植的根茎类蔬菜，具有很高的经济价值。胡萝卜根和地上器官的香气主要由萜烯定义。我们发现橙色胡萝卜品种的叶子也会产生大量的苯丙烯甲基丁香酚和甲基异丁香酚。值得注意的是，甲基丁香酚在幼叶中含量最高，而甲基异丁香酚是成熟叶组织中的主要苯丙烯。本研究的目的是阐明这些化合物的生物合成和积累的生物化学和分子生物学。使用可用的基因组和转录组数据，我们分离出编码丁香酚/异丁香酚合酶 (DcE(I)GS1) 的 cDNA，这是一种 NADPH 依赖性酶，可将醋酸松柏酯转化为丁香酚。这种酶表现出双重产品特异性，并产生丙烯基苯酚异丁香酚和烯丙基苯酚丁香酚。此外，我们鉴定了一种编码 S-腺苷-L-甲硫氨酸：丁香酚/异丁香酚 O-甲基转移酶 1 (DcE(I)OMT1) 的 cDNA，它通过甲基化各自前体的对羟基基团产生甲基丁香酚和甲基异丁香酚。DcE(I)GS1 和 DcE(I)OMT1 在种子、根、幼叶和成熟叶中均有表达，DcE(I)OMT1 转录水平在叶片中最高。DcE(I)GS1 蛋白与八角茴香醇/异丁香酚合酶有 67% 的相同性，对于醋酸松柏酯的表观 Km 为 247 μM。DcEOMT1 对丁香酚的催化效率是异丁香酚的五倍以上，丁香酚的 Km 值为 40 μM，异丁香酚的 Km 值为 115 μM。