Ro Dae-Kyun, Bohlmann Jörg
Michael Smith Laboratories, and Departments of Botany and Forest Science, University of British Columbia, 321-2185 East Mall, Vancouver, BC, Canada.
Phytochemistry. 2006 Aug;67(15):1572-8. doi: 10.1016/j.phytochem.2006.01.011. Epub 2006 Feb 23.
Diterpene resin acids are prominent defense compounds against insect pests and pathogens in conifers. Biochemical and molecular analyses in grand fir (Abies grandis), Norway spruce (Picea abies), and loblolly pine (Pinus taeda) have identified two classes of genes and enzymes that generate much of the structural diversity of terpenoid defense compounds: The terpenoid synthases (TPS) and cytochrome P450 monooxgenases (P450). Using a single substrate, geranylgeranyl diphosphate, families of single-product and multi-product diterpene synthases generate an array of cyclic diterpene olefins. These diterpenes are converted to diterpene resin acids by activity of one or more P450 enzymes. A few conifer diterpene synthases have previously been cloned and characterized in grand fir and in Norway spruce. We have also previously shown that the loblolly pine P450 abietadienol/abietadienal oxidase (PtAO) catalyzes multiple oxidations of several diterpene alcohols and aldehydes. Conifer diterpene synthases are thought to function in plastids while P450s can also be localized to plastids or to the endoplasmic reticulum (ER). Here, we show that a loblolly pine cDNA (PtTPS-LAS) encodes a typical multi-product conifer diterpene synthase that forms levopimaradiene, abietadiene, palustradiene, and neoabietadiene similar to the grand fir abietadiene synthase and Norway spruce levopimaradiene/abietadiene synthase. Subcellular targeting of PtTPS-LAS and PtAO to plastids and ER, respectively, was shown with green fluorescent fusion protein expression in tobacco cells. These data suggest that enzymes for conifer diterpene resin acid biosynthesis are localized to at least two different subcellular compartments, plastids and ER, requiring efficient transport of intermediates and secretion of diterpene resin acids into the extracelluar space.
二萜树脂酸是针叶树抵御害虫和病原体的重要防御化合物。对巨冷杉（Abies grandis）、挪威云杉（Picea abies）和火炬松（Pinus taeda）进行的生化和分子分析，已鉴定出两类基因和酶，它们产生了萜类防御化合物的大部分结构多样性：萜类合酶（TPS）和细胞色素P450单加氧酶（P450）。单产物和多产物二萜合酶家族利用单一底物香叶基香叶基二磷酸，生成一系列环状二萜烯烃。这些二萜通过一种或多种P450酶的活性转化为二萜树脂酸。此前已在巨冷杉和挪威云杉中克隆并表征了一些针叶树二萜合酶。我们之前还表明，火炬松P450枞二烯醇/枞二烯醛氧化酶（PtAO）催化几种二萜醇和醛的多次氧化。针叶树二萜合酶被认为在质体中发挥作用，而P450也可以定位于质体或内质网（ER）。在这里，我们表明，火炬松cDNA（PtTPS-LAS）编码一种典型的多产物针叶树二萜合酶，该酶形成左旋海松二烯、枞二烯、沼泽二烯和新枞二烯，类似于巨冷杉枞二烯合酶和挪威云杉左旋海松二烯/枞二烯合酶。在烟草细胞中通过绿色荧光融合蛋白表达，分别显示了PtTPS-LAS和PtAO在质体和内质网中的亚细胞定位。这些数据表明，针叶树二萜树脂酸生物合成的酶定位于至少两个不同的亚细胞区室，即质体和内质网，这需要中间体的有效运输以及二萜树脂酸分泌到细胞外空间。
