Abstract

There are several causes for the great diversity in floral terpenes. The terpene products are determined by the catalytic fidelity, efficiency and plasticity of the active sites of terpene synthases (TPSs). However, the molecular mechanism of TPS in catalyzing terpene biosynthesis and its evolutionary fate in wild plant species remain largely unknown. In this study, the functionality of terpene synthases and their natural variants were assessed in two Northeastern Asia endemic columbine species and their natural hybrid. Synoptically, TPS7, TPS8, and TPS9 were highly expressed in these Aquilegia species from the Zuojia population. The in vitro and in vivo enzymatic assays revealed that TPS7 and TPS8 mainly produced (+)-limonene and β-sesquiphellandrene, respectively, whereas TPS9 produced pinene, similar to the major components released from Aquilegia flowers. Multiple sequence alignment of Aquilegia TPS7 and TPS8 in the Zuojia population revealed amino acid polymorphisms. Domain swapping and amino acid substitution assays demonstrated that 413A, 503I and 529D had impacts on TPS7 catalytic activity, whereas 420G, 538F and 545 L affected the ratio of β-sesquiphellandrene to β-bisabolene in TPS8. Moreover, these key polymorphic amino acid residues were found in Aquilegia species from the Changbai Mountain population. Interestingly, amino acid polymorphisms in TPSs were present in individuals with low expression levels, and nonsynonymous mutations could impact the catalytic activity or product specificity of these genes. The results of this study will shed new light on the function and evolution of TPS genes in wild plant species and are beneficial to the modification of plant fragrances.

中文翻译：

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东北亚野生耧斗菜花中萜合酶变异的表征

摘要

花萜的多样性有几个原因。萜烯产物由萜烯合酶 (TPS) 活性位点的催化保真度、效率和可塑性决定。然而，TPS催化萜烯生物合成的分子机制及其在野生植物物种中的进化命运仍然很大程度上未知。在这项研究中，在两种东北亚特有的鸽子物种及其天然杂种中评估了萜烯合酶及其天然变体的功能。从表面上看，TPS7、TPS8和TPS9在来自左家种群的这些耧斗菜属物种中高度表达。体外和体内_酶促测定显示，TPS7和TPS8分别主要产生 (+)-柠檬烯和 β-倍半水芹烯，而TPS9产生蒎烯，类似于从耧斗花中释放的主要成分。左家种群中耧斗菜TPS7 和 TPS8 的多序列比对揭示了氨基酸多态性。结构域交换和氨基酸取代分析表明，413A、503I 和 529D 对 TPS7 催化活性有影响，而 420G、538F 和 545 L 影响 TPS8 中 β-倍半水芹烯与 β-红没药烯的比例。此外，这些关键的多态性氨基酸残基在耧斗菜中发现长白山种群中的物种。有趣的是，TPS 中的氨基酸多态性存在于低表达水平的个体中，非同义突变可能会影响这些基因的催化活性或产物特异性。本研究结果将为野生植物物种中TPS基因的功能和进化提供新的启示，有利于植物香味的修饰。