Younkin Gordon C, Alani Martin L, Züst Tobias, Jander Georg
Boyce Thompson Institute, Ithaca, New York 14853.
Plant Biology Section, School of Integrative Plant Science, Cornell University, Ithaca, New York 14853.
bioRxiv. 2024 Apr 11:2024.04.10.588904. doi: 10.1101/2024.04.10.588904.
Plants commonly produce families of structurally related metabolites with similar defensive functions. This apparent redundancy raises the question of underlying molecular mechanisms and adaptive benefits of such chemical variation. Cardenolides, a class defensive compounds found in the wallflower genus (L., Brassicaceae) and scattered across other plant families, show substantial structural variation, with glycosylation and hydroxylation being common modifications of a steroid core, which itself may vary in terms of stereochemistry and saturation. Through a combination of chemical mutagenesis and analysis of gene coexpression networks, we identified four enzymes involved in cardenolide biosynthesis in that work together to determine stereochemistry at carbon 5 of the steroid core: Ec3βHSD, a 3β-hydroxysteroid dehydrogenase, Ec3KSI, a ketosteroid isomerase, EcP5βR2, a progesterone 5β-reductase, and EcDET2, a steroid 5α-reductase. We biochemically characterized the activity of these enzymes and generated CRISPR/Cas9 knockout lines to confirm activity . Cardenolide biosynthesis was not eliminated in any of the knockouts. Instead, mutant plants accumulated cardenolides with altered saturation and stereochemistry of the steroid core. Furthermore, we found variation in carbon 5 configuration among the cardenolides of 44 species of , where the occurrence of some 5β-cardenolides is associated with the expression and sequence of P5βR2. This may have allowed species to fine-tune their defensive profiles to target specific herbivore populations over the course of evolution.
Plants use an array of toxic compounds to defend themselves from attack against insects and other herbivores. One mechanism through which plants may evolve more toxic compounds is through modifications to the structure of compounds they already produce. In this study, we show how plants in the wallflower genus use four enzymes to fine-tune the structure of toxic metabolites called cardenolides. Natural variation in the sequence and expression of a single enzyme called progesterone 5β-reductase 2 partly explains the variation in cardenolides observed across the genus. These alterations to cardenolide structure over the course of evolution suggests that there may be context-dependent benefits to to invest in one cardenolide variant over another.
植物通常会产生结构相关且具有相似防御功能的代谢物家族。这种明显的冗余引发了关于这种化学变异的潜在分子机制和适应性益处的问题。强心甾内酯是一类在桂竹香属（十字花科）中发现并分散于其他植物科的防御性化合物，其结构存在显著变异，糖基化和羟基化是甾体核心常见的修饰方式，而甾体核心本身在立体化学和饱和度方面也可能有所不同。通过化学诱变和基因共表达网络分析相结合的方法，我们在桂竹香中鉴定出了四种参与强心甾内酯生物合成的酶，它们共同作用以确定甾体核心C-5位的立体化学结构：Ec3βHSD，一种3β-羟基甾体脱氢酶；Ec3KSI，一种酮甾体异构酶；EcP5βR2，一种孕酮5β-还原酶；以及EcDET2，一种甾体5α-还原酶。我们对这些酶的活性进行了生化特性分析，并构建了CRISPR/Cas9基因敲除系以确认其活性。在任何一个敲除系中，强心甾内酯的生物合成均未被消除。相反，突变植株积累了甾体核心饱和度和立体化学结构发生改变的强心甾内酯。此外，我们发现桂竹香属44个物种的强心甾内酯中C-5位构型存在差异，其中一些5β-强心甾内酯的出现与P5βR2的表达和序列有关。这可能使桂竹香属物种在进化过程中能够微调其防御特征，以针对特定的食草动物种群。
植物利用一系列有毒化合物来抵御昆虫和其他食草动物的攻击。植物可能进化出更具毒性化合物的一种机制是通过对它们已经产生的化合物结构进行修饰。在本研究中，我们展示了桂竹香属植物如何利用四种酶来微调称为强心甾内酯的有毒代谢物的结构。一种名为孕酮5β-还原酶2的单一酶的序列和表达的自然变异部分解释了在桂竹香属中观察到的强心甾内酯的变异。在进化过程中，强心甾内酯结构的这些改变表明，对于桂竹香属而言，投资于一种强心甾内酯变体而非另一种变体可能存在依赖于环境的益处。
