Fujita M, Gang D R, Davin L B, Lewis N G
Institute of Biological Chemistry, Washington State University, Pullman, Washington 99164-6340, USA.
J Biol Chem. 1999 Jan 8;274(2):618-27. doi: 10.1074/jbc.274.2.618.
Although the heartwood of woody plants represents the main source of fiber and solid wood products, essentially nothing is known about how the biological processes leading to its formation are initiated and regulated. Accordingly, a reverse transcription-polymerase chain reaction-guided cloning strategy was employed to obtain genes encoding pinoresinol-lariciresinol reductases from western red cedar (Thuja plicata) as a means to initiate the study of its heartwood formation. (+)-Pinoresinol-(+)-lariciresinol reductase from Forsythia intermedia was used as a template for primer construction for reverse transcription-polymerase chain reaction amplifications, which, when followed by homologous hybridization cloning, resulted in the isolation of two distinct classes of putative pinoresinol-lariciresinol reductase cDNA clones from western red cedar. A representative of each class was expressed as a fusion protein with beta-galactosidase and assayed for enzymatic activity. Using both deuterated and radiolabeled (+/-)-pinoresinols as substrates, it was established that each class of cDNA encoded a pinoresinol-lariciresinol reductase of different (opposite) enantiospecificity. Significantly, the protein from one class converted (+)-pinoresinol into (-)-secoisolariciresinol, whereas the other utilized the opposite (-)-enantiomer to give the corresponding (+)-form. This differential substrate specificity raises important questions about the role of each of these individual reductases in heartwood formation, such as whether they are expressed in different cells/tissues or at different stages during heartwood development.
虽然木本植物的心材是纤维和实木产品的主要来源，但对于导致其形成的生物学过程是如何启动和调控的，人们几乎一无所知。因此，采用了逆转录-聚合酶链反应引导的克隆策略，从西部红雪松（Thuja plicata）中获取编码松脂醇-落叶松脂醇还原酶的基因，以此作为启动其心材形成研究的一种手段。来自连翘的（+）-松脂醇-（+）-落叶松脂醇还原酶被用作逆转录-聚合酶链反应扩增引物构建的模板，随后通过同源杂交克隆，从西部红雪松中分离出了两类不同的假定松脂醇-落叶松脂醇还原酶cDNA克隆。每类的一个代表被表达为与β-半乳糖苷酶的融合蛋白，并对其酶活性进行了测定。使用氘代和放射性标记的（±）-松脂醇作为底物，确定每类cDNA编码一种具有不同（相反）对映体特异性的松脂醇-落叶松脂醇还原酶。值得注意的是，一类蛋白将（+）-松脂醇转化为（-）-开环异落叶松脂醇，而另一类则利用相反的（-）-对映体生成相应的（+）-形式。这种不同的底物特异性引发了关于这些单个还原酶在心材形成中各自作用的重要问题，例如它们是否在不同的细胞/组织中表达，或者在心材发育的不同阶段表达。
