Revealing the role of autotoxicity protection for defense metabolism innovations in Solanaceae using a multiomics approach

许多植物特化代谢物（PSM）具有防御功能，取消特定的途径步骤可导致有毒中间体的积累，这表明除了基于防御的功能选择外，自毒性避免在PSM生物化学进化中起着关键作用。然而，我们目前还不知道自毒性保护机制在多大程度上以及如何与防御功能一起进化。这些具有挑战性的问题需要多物种和多器官的方法整合基因和PSM水平的信息（多组学图谱）来测试，使用功能和进化研究，避免自毒性驱动进化的三个关键预测：1)参与自毒性预防的基因应该检测到PSM途径的积极选择的强烈特征；2)这些基因的表达可能在与细胞生长和/或分化相关的特定细胞类型中富集；3)在具有特定PSM创新的近亲物种中，应该检测到类似的器官水平的选择特征，因为由内在细胞功能施加的选择可能不如生态选择那么动态。在这里，通过我们最近在烟草中二萜苷（DTG）的生物合成和（自）毒性机制方面的突破，以及其他小组在甾体糖生物碱（SGA）的生物合成方面的工作，两种突出的茄科PSM防御，我们将实现最先进的计算代谢组学，转录组学，单细胞基因组学和遗传操作工具的整合，以应对这一挑战。我们的目标是(i)建立8种茄属植物的代谢组学-转录组学综合图谱，以确定DTG和SGA途径的生化创新；（ii）回溯这些途径的分子进化，并表征具有强正选择特征的基因；（iii）作为案例研究，利用单细胞基因组学和遗传操作的结合，从功能上破译了SGAs进化中自毒性避免的贡献。这项工作的结果将使我们能够评估自毒性预防是否是驱动茄科化学多样化的关键选择压力。更广泛地说，EVOMET将解决我们对植物防御进化理解的范式转变，为下一代作物保护策略的设计提供关键见解，并在植物进化生物学中实现新的比较单细胞测序工具。

Many plant specialized metabolites (PSMs) function as defenses and abrogating particular pathway steps can lead to the accumulation of toxic intermediates, indicating that in addition to defense-based functional selection, autotoxicity avoidance plays a key role in the evolution of the PSM biochemistry. However, we currently do not know to which extent and how autotoxicity protection mechanisms evolve in concert with defensive functions. These challenging questions require a multi-species and -organ approach integrating gene -and PSM-level information (multiomics atlases) to test, using both functional and evolutionary studies, three key predictions of autotoxicity avoidance-driven evolution: that 1) strong signatures of positive selection for a PSM pathway should be detected for genes participating in autotoxicity prevention; 2) expression of those genes should likely be enriched in specific cell types associated with cell growth and/or differentiation; and that 3) similar organ-level signatures of selection should be detected among closely-related species harboring a given PSM innovation, since the selection imposed by intrinsic cellular functions is likely to be less dynamic than ecological selections. Here, by building on our recent breakthrough on the biosynthesis and (auto)toxicity mechanisms of diterpene glycosides (DTG) in tobacco as well as on other groups’ work on the biosynthesis of steroidal glycoalkaloids (SGA), two prominent Solaneaceae PSM defenses, we will implement a state-of-the-art integration of computational metabolomics, transcriptomics, single-cell genomics and genetic manipulation tools to address this challenge. We aim to (i) establish integrated metabolomics-transcriptomics atlases for eight solanaceous species to pinpoint on biochemical innovations in the DTG and SGA pathways; (ii) retrace the molecular evolution of these pathways and characterize genes with signatures of strong positive selection; and (iii), as a case-study, functionally decipher the contribution of autotoxicity avoidance for SGAs’ evolution using a combination of single-cell genomics and genetic manipulations. The outcomes of this work will allow us to evaluate whether autotoxicity prevention is a key selection pressure driving the chemodiversification in Solanaceae. More broadly, EVOMET will address a paradigm shift in our understanding of plant defense evolution, provide key insights for the design of next-generation crop protection strategies, and implement new comparative single-cell sequencing tools in plant evolutionary biology.