RNAi Suppression of Arogenate Dehydratase1 Reveals That Phenylalanine Is Synthesized Predominantly via the Arogenate Pathway in Petunia Petals
RNAi Suppression of Arogenate Dehydratase1 Reveals That Phenylalanine Is Synthesized Predominantly via the Arogenate Pathway in Petunia Petals
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DOI：
10.1105/tpc.109.073247
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发表时间：
2010-03-01
期刊：
影响因子：
11.6
通讯作者：
Dudareva, Natalia
中科院分区：
文献类型：
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作者：
Maeda, Hiroshi;Shasany, Ajit K.;Dudareva, Natalia
L-苯丙氨酸是一种蛋白质构建基团，是多种酚类化合物的前体，由苯丙酮酸和芳香酸脱水酶合成，其中芳香酸脱水酶和苯丙酮酸脱水酶分别起关键作用。在这里，我们使用矮牵牛花，它富含苯丙氨酸衍生的挥发性物质，以确定参与植物苯丙氨酸形成的生物合成途径。在已鉴定的3个矮牵牛ADT中，ADT1在矮牵牛花瓣中的表达水平最高，并且在整个花发育过程中与内源Phe水平呈正相关。ADT1对芳香酸表现出严格的底物专一性，但在三种ADT中催化效率最低。在矮牵牛花瓣中，通过RNA干扰抑制ADT1显著降低了ADT活性、Phe水平和下游的苯丙烷/苯类挥发物。意想不到的是，转基因花瓣中的草原酸水平没有变化，而莽草酸和色氨酸的水平却降低了。稳定同位素标记实验表明，ADT1的抑制导致了向莽草酸的碳通量的下调。然而，外源的莽草酸供应绕过了这一负面调控，导致了无机酸积累的增加。在转基因花瓣中取食莽草酸还导致了苯基丙酮酸和苯丙酮酸的积累，部分恢复了降低的苯丙酮酸水平，这表明苯丙酮酸途径也可以在植物中运行。这些结果提供了遗传证据，证明Phe主要是通过矮牵牛花瓣中的无源酸合成的，并揭示了莽草酸途径的一种新的转录后调节。
L-Phe, a protein building block and precursor of numerous phenolic compounds, is synthesized from prephenate via an arogenate and/or phenylpyruvate route in which arogenate dehydratase (ADT) or prephenate dehydratase, respectively, plays a key role. Here, we used Petunia hybrida flowers, which are rich in Phe-derived volatiles, to determine the biosynthetic routes involved in Phe formation in planta. Of the three identified petunia ADTs, expression of ADT1 was the highest in petunia petals and positively correlated with endogenous Phe levels throughout flower development. ADT1 showed strict substrate specificity toward arogenate, although with the lowest catalytic efficiency among the three ADTs. ADT1 suppression via RNA interference in petunia petals significantly reduced ADT activity, levels of Phe, and downstream phenylpropanoid/benzenoid volatiles. Unexpectedly, arogenate levels were unaltered, while shikimate and Trp levels were decreased in transgenic petals. Stable isotope labeling experiments showed that ADT1 suppression led to downregulation of carbon flux toward shikimic acid. However, an exogenous supply of shikimate bypassed this negative regulation and resulted in elevated arogenate accumulation. Feeding with shikimate also led to prephenate and phenylpyruvate accumulation and a partial recovery of the reduced Phe level in transgenic petals, suggesting that the phenylpyruvate route can also operate in planta. These results provide genetic evidence that Phe is synthesized predominantly via arogenate in petunia petals and uncover a novel posttranscriptional regulation of the shikimate pathway.