Defining the Tyrosine Biosynthetic Pathways in Plants

非技术摘要：植物合成一种芳香族氨基酸酪氨酸(Tyr)，它是人类饮食中的一种基本营养物质，也是许多植物专用化合物的关键前体。这些来源于Tyr的植物天然产物对植物的生长、发育和环境反应具有深远的影响，对人类也具有重要的营养和药理活性(如维生素E、吗啡)。尽管它们在植物和人类生物学中都很重要，但我们仍然不知道Tyr是如何在植物中合成的。因此，该项目将获得基础知识，以提高农作物和药用植物的营养质量或药用价值，并有可能改善植物生长和胁迫反应，以可持续地生产粮食和生物质。一项名为“色素艺术”的推广活动将利用从芳香氨基酸(例如甜菜碱)中提取的诱人和有益的植物色素。虽然年纪较小的孩子会欣赏天然色素中美丽而复杂的颜色，但父母和年龄较大的孩子将有机会了解这些色素是如何由植物合成的。项目技术简介：越来越多的证据表明，许多植物通过中间体芳香酸合成酪氨酸，因此不同于通常使用4-羟基苯丙酮酸作为中间体的微生物模型。然而，不同植物物种之间的Tyr生物合成途径似乎存在显著差异。该项目的目标是确定植物体内酪氨酸的生物合成途径(S)，并确定它们的区划和关键的调控步骤(S)。这项拟议的研究将建立在Pi最近分离的拟南芥和豆类基因的基础上，这些基因编码酪氨酸途径的关键酶--阿罗酸和预苯酸脱氢酶(ADH和PDH)。拟南芥ADHS对芳香酸具有很强的底物专一性，定位于质体中，并被Tyr抑制。相比之下，豆类脱氢酶专门使用预苯酸，缺乏叶绿体转运肽，并且对Tyr不敏感。本研究的基本假设是拟南芥主要通过酪氨酸调控的胞质芳香酸途径合成酪氨酸，而豆科植物则通过芳香酸和4-羟基苯丙酮酸合成酪氨酸，其定位和调控机制尚不清楚。在这个项目中，首先计划对新发现的豆科植物多环芳烃进行动力学分析和本地化研究(目标1)。在植物中，PDHs和ADHS的功能将用两个不同的植物模型--紫花苜蓿(Aim 2)和拟南芥(Aim 3)--进行研究和比较。将在产生的突变株和转基因株中进一步进行靶向代谢物分析和稳定同位素喂养实验，以直接测量碳流在替代Tyr途径中的分布。虽然初级新陈代谢通常被认为在植物物种中是保守的，但我们的初步数据表明并非如此。该项目将直接检验这一假设，并解决酪氨酸生物合成这一主要代谢途径如何在不同的植物中进化，以支持下游专门代谢中存在的巨大化学多样性。

Non-technical summary:Plants synthesize an aromatic amino acid tyrosine (Tyr), which is an essential nutrient in human diets and a key precursor of numerous plant specialized compounds. These Tyr-derived plant natural products have profound impacts on plant growth, development and environmental responses, and also have important nutritional and pharmacological activities in humans (e.g., vitamin E, morphine). Despite their importance in both plant and human biology, we still do not know how Tyr is synthesized in plants. Therefore, this project will gain fundamental knowledge to enhance the nutritional quality or pharmaceutical values of crops and medicinal plants, and can potentially improve plant growth and stress responses for sustainable production of food and biomass. An outreach event, "Pigment-Art", will take advantage of attractive and beneficial plant pigments derived from aromatic amino acids (e.g., betalains). While younger children will appreciate the beautiful and complex colors in natural pigments, parents and older children will have opportunities to learn about how these pigments are synthesized by plants. Technical Description of the Project:Accumulating evidence suggests that many plants synthesize Tyr via the intermediate arogenate and therefore differ from microbial models that usually use 4-hydroxyphenylpyruvate as an intermediate. However, significant variations appear to exist for the Tyr biosynthetic routes among different plant species. The goals of this project are to define the Tyr biosynthetic route(s) in plants and determine their compartmentalization and crucial regulatory step(s). The proposed research will build upon PI's recent isolation of Arabidopsis and legume genes encoding arogenate and prephenate dehydrogenases (ADH and PDH), key enzymes of the Tyr pathway. Arabidopsis ADHs have strong substrate specificity toward arogenate, are localized in the plastids and inhibited by Tyr. In contrast, legume dehydrogenases specifically use prephenate, lack a plastid transit peptide, and are insensitive to Tyr. The primary hypothesis of this study is that Arabidopsis mainly uses the plastidic arogenate pathway that is regulated by Tyr, whereas legumes synthesize Tyr via both arogenate and 4-hydroxyphenylpyruvate with unknown localization and regulatory mechanism. In this project, kinetic analysis and localization studies are first planned for the newly identified legume PDHs (Aim 1). In planta functions of PDHs and ADHs will be investigated and compared using two contrasting plant models, Medicago truncatula (Aim 2) and Arabidopsis thaliana (Aim 3). Targeted metabolite profiling and stable-isotope feeding experiments will be further conducted in the generated mutants and transgenics to directly measure the distribution of carbon flow among the alternative Tyr pathways. Although primary metabolism is generally assumed to be conserved across plant species, our preliminary data indicate otherwise. This project will directly test this assumption and address how Tyr biosynthesis, a primary metabolic pathway, has evolved in different plants to support the enormous chemical diversity that exists in the downstream specialized metabolism.